Anti-endoglin antibodies and uses thereof

ABSTRACT

The present application relates to compositions of humanized and deimmunized anti-endoglin antibodies and antigen-binding fragments thereof. One aspect relates to antibodies having one or more modifications in at least one amino acid residue of at least one of the framework regions of the variable heavy chain, the variable light chain or both. Another aspect relates to anti-endoglin antibodies which inhibit or treat fibrosis.

CROSS-REFERENCE

This application is a continuation-in-part of International ApplicationNo. PCT/US15/60136, filed Nov. 11, 2015, which claims the benefit ofU.S. Provisional Application No. 62/078,788, filed Nov. 12, 2014, whichapplication is incorporated herein by reference in its entirety; andalso claims the benefit of U.S. Provisional Application No. 62/161,669,filed May 14, 2015, which application is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

Endoglin, also known as, inter alia, CD105 or edg-1, is a type Ihomodimeric membrane glycoprotein which is expressed at high levels inproliferating vascular endothelial cells (Burrows et al., 1995, Clin.Cancer Res. 1:1623-1634). However, there is some expression of endoglinby the vascular endothelium of normal tissues (Burrows et al., Id; Wanget al., 1993, Int. J. Cancer 54:363-370). Human endoglin is known tospecifically bind transforming growth factor-β (TGF-β), and the deducedamino acid sequence of endoglin has strong homology to β-glycan, a typeof TGF-β receptor.

Several anti-endoglin antibodies, in particular anti-endoglin monoclonalantibodies (“mAb”), have been described. mAb SN6j is a monoclonalantibody generated from immunization of mice with glycoprotein mixturesof cell membranes of human leukemia cells (Haruta and Seon, 1986, Proc.Natl. Acad. Sci. USA 83:7898-7902). SN6 is a murine mAb that recognizeshuman endoglin. C-SN6j is a chimeric form of SN6j which has mousevariable regions and a human constant region. mAb 44G4 is an antibodygenerated from immunization of mice with whole cell suspensions of humanpre-B leukemia cells (Gougos and Letarte, 1988, J. Immunol.141:1925-1933; 1990, J. Biol. Chem. 265:8361-8364). 44G4 is also amurine mAb that recognizes human endoglin. mAb MJ7/18 is an antibodygenerated from immunization of rats with inflamed mouse skins (Ge andButcher, 1994, Id). MJ7/18 is a mAb that recognizes murine endoglin. mAbTec-11 is an antibody generated from immunization of mice with humanumbilical vein endothelial cells (Burrows et al., 1995, Clin. CancerRes. 1:1623-1634). Tec-11 is a murine mAb with reactivity restricted tohuman endoglin (Burrows et al., Clin. Cancer Res., 1995; 1(12):1623-34).

SUMMARY OF THE INVENTION

Provided herein are humanized and deimmunized antibodies orantigen-binding fragments thereof that bind to endoglin. Such antibodieshave uses in purification, detection, diagnostic and therapeuticapplications. Also provided herein are humanized and deimmunizedantibodies or antigen-binding fragments thereof that bind to one or morespecies or variants of endoglin and treat, inhibit, or amelioratefibrosis.

The humanized and deimmunized antibodies and antigen-binding fragmentswhich bind endoglin and are described herein can be used to treat,inhibit, or ameliorate fibrosis including, but not limited to,pulmonary, liver, cardiac, or kidney fibrosis. The humanized anddeimmunized antibodies and antigen-binding fragments described hereincan also be used in medicaments for the treatment, inhibition, oramelioration of fibrosis.

Provided herein are humanized and deimmunized antibodies thatspecifically bind to endoglin. Also provided herein are humanized anddeimmunized antibodies that specifically bind to endoglin to preventbinding by the endoglin ligand BMP. Also provided herein are humanizedand deimmunized antibodies that specifically bind to endoglin andinhibit downstream signaling of the Smad 1/5/8 pathway.

Also provided herein are antibodies of an IgG1 isotype. Also providedherein are antibodies of an IgG4 isotype. Also provided herein areantibodies of an IgG2 isotype.

Provided herein are antibodies to endoglin that inhibit the BMP9signaling pathway and interrupt SMAD 1/5/8 signaling in fibroblasts toinhibit fibrosis.

Provided herein are methods for treating, inhibiting, or amelioratingfibrosis comprising administering a composition comprising a humanizedand deimmunized antibody or antigen-binding fragment described hereinthat binds to endoglin associated with the disease or disorder andprevents fibrosis (i.e., preventing, treating, ameliorating, orlessening the severity of fibrosis).

Provided herein are methods for treating, inhibiting, or amelioratingtreating fibrosis, comprising administering a composition comprising ahumanized and deimmunized antibody or antigen-binding fragment describedherein that binds to endoglin treats fibrosis.

Provided herein are methods for treating, inhibiting, or amelioratingtreating fibrosis, comprising administering a composition comprising ahumanized and deimmunized antibody or antigen-binding fragment describedherein that binds to a endoglin receptor and blocks BMP9 binding toendoglin.

Provided herein are methods for treating, inhibiting, or amelioratingtreating fibrosis, comprising administering a composition comprising ahumanized and deimmunized antibody or antigen-binding fragment describedherein that binds to an endoglin receptor and inhibits Smad 1/5/8signaling.

Provided herein is a method of treating or inhibiting fibrosis in asubject in need thereof, comprising administering to the subject acomposition comprising an antibody, or antigen-binding fragment thereof,comprising a heavy chain variable region having an amino acid sequenceset forth as SEQ ID NO: 89 and a light chain variable region having anamino acid sequence set forth as SEQ ID NO: 93 and a pharmaceuticallyacceptable excipient, whereby fibrosis is treated or inhibited.

Provided herein is a method of treating or inhibiting fibrosis in asubject in need thereof, comprising administering to the subject acomposition comprising an antibody, or antigen-binding fragment thereof,that binds endoglin, comprising a heavy chain variable region having anamino acid sequence set forth as SEQ ID NO: 89 and a light chainvariable region having an amino acid sequence set forth as SEQ ID NO:93, wherein: said heavy chain variable region further comprises one ormore modifications selected from the group consisting of a substitutionof glycine (G) by alanine (A) or serine (S) at position 49; asubstitution of alanine (A) by isoleucine (I) at position 51; asubstitution of lysine (K) by arginine (R) or asparagine (Q) at position52b; a substitution of leucine (L) by valine (V) at position 78utilizing the Kabat numbering system; and the light chain variableregion further comprises one or more modifications selected from thegroup consisting of a substitution of methionine (M) by leucine (L) atposition 4; a substitution of alanine (A) by valine (V) at position 19;a substitution of threonine (T) by serine (S) at position 22; asubstitution of alanine (A) by isoleucine (I) at position 48; and asubstitution of threonine (T) by serine (S) at position 51 utilizing theKabat numbering system; and a pharmaceutically acceptable excipient,whereby fibrosis is treated or inhibited.

In such methods, the antibody, or antigen-binding fragment thereof, cancomprise a heavy chain variable region having an amino acid sequence setforth as SEQ ID NO: 88, 89, 90, 91 or 92; and a light chain variableregion having an amino acid sequence set forth as SEQ ID NO: 93, 94, 95,96, 97, 100, 102, or 103.

In such methods, the antigen-binding fragment can be a Fab fragment, aFab′ fragment, a F(ab′)₂ fragment, an Fv fragment, an scFv fragment, asingle chain binding polypeptide, a Fd fragment, a variable heavy chain,a variable light chain or a dAb fragment.

The antibody, or antigen-binding fragment thereof, can be furtherlabeled with a therapeutic label.

In one aspect, fibrosis is liver fibrosis. Liver fibrosis includes, butis not limited to, cirrhosis and associated conditions such as chronicviral hepatitis, non-alcoholic fatty liver disease (NAFLD), alcoholicsteatohepatitis (ASH), non-alcoholic steatohepatitis (NASH), primarybiliary cirrhosis (PBC), biliary cirrhosis, autoimmune hepatitis.

In some instances, liver fibrosis can be caused by a chronic insult tothe liver from a parasite or a virus. A parasite or virus can be, forexample, Hepatitis B Virus (HBV), Hepatitis B Virus (HCV), HumanImmunodeficiency Virus (HIV), or schistosomiasis.

In another aspect, fibrosis is kidney fibrosis. Kidney fibrosis can becaused by chronic kidney disease, metabolic syndrome, vesicoureteralreflux, tubulointerstitial renal fibrosis, diabetes, glomerulonephritisor glomerular nephritis (GN), focal segmental glomerulosclerosis andmembranous glomerulonephritis, or mesangiocapillary GN.

In another aspect, fibrosis is pulmonary fibrosis. Pulmonary fibrosisincludes, but is not limited to idiopathic pulmonary fibrosis (IPF) orcryptogenic fibrosing alveolitis, chronic fibrosing interstitialpneumonia, interstitial lung disease (ILD), diffuse parenchymal lungdisease (DPLD), idiopathic interstitial pneumonia, acute respiratorydistress syndrome (ARDS).

In another aspect, fibrosis is a neurofibromatosis. Neurofibromatosisincludes, but is not limited to, a neurofibromatosis type 1 and aneurofibromatosis type 2. In one instance, neurofibromatosis is aneurofibromatosis type 1. In another instance, neurofibromatosis is aneurofibromatosis type 2.

In another aspect, fibrosis is myelofibrosis.

Any of the methods described herein may further comprise administeringone or more fibrosis inhibitors.

In some instances, the anti-endoglin antibody, or antigen-bindingfragment and one or more fibrosis inhibitors are administered at thesame site.

In other instances, the anti-endoglin antibody, or antigen-bindingfragment and one or more fibrosis inhibitors are administered atdifferent sites.

In other instances, the anti-endoglin antibody, or antigen-bindingfragment and one or more fibrosis inhibitors are administeredsequentially.

In other instances, the anti-endoglin antibody, or antigen-bindingfragment and one or more fibrosis inhibitors are administeredconcurrently.

An antibody or antigen-binding fragment thereof can be administered inan amount of about 0.01 mg/kg, about 0.05 mg/kg, about 0.1 mg/kg, about0.5 mg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 20 mg/kg,about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 60 mg/kg, about 70mg/kg, about 80 mg/kg, about 90 mg/kg, about 100 mg/kg, about 125 mg/kg,about 150 mg/kg, about 175 mg/kg, or about 200 mg/kg per patient.

Provided herein is a method of inhibiting BMP signaling comprisingcontacting cells with an antibody, or antigen-binding fragment thereof,wherein the antibody, or antigen-binding fragment thereof, comprises aheavy chain variable region having an amino acid sequence set forth asSEQ ID NO: 89 and a light chain variable region having an amino acidsequence set forth as SEQ ID NO: 93.

Provided herein is a method of inhibiting BMP signaling in fibroblastscomprising contacting cells with an antibody, or antigen-bindingfragment thereof, wherein the antibody, or antigen-binding fragmentthereof, comprises a heavy chain variable region having an amino acidsequence set forth as SEQ ID NO: 89 and a light chain variable regionhaving an amino acid sequence set forth as SEQ ID NO: 93, wherein saidheavy chain variable region further comprises one or more modificationsselected from the group consisting of a substitution of glycine (G) byalanine (A) or serine (S) at position 49; a substitution of alanine (A)by isoleucine (I) at position 51; a substitution of lysine (K) byarginine (R) or asparagine (Q) at position 52b; a substitution ofleucine (L) by valine (V) at position 78 utilizing the Kabat numberingsystem; and the light chain variable region further comprises one ormore modifications selected from the group consisting of a substitutionof methionine (M) by leucine (L) at position 4; a substitution ofalanine (A) by valine (V) at position 19; a substitution of threonine(T) by serine (S) at position 22; a substitution of alanine (A) byisoleucine (I) at position 48; and a substitution of threonine (T) byserine (S) at position 51 utilizing the Kabat numbering system, whereinBMP signaling is inhibited.

In such methods, an antibody, or antigen-binding fragment thereof, cancomprise a heavy chain variable region having an amino acid sequence setforth as SEQ ID NO: 88, 89, 90, 91 or 92; and a light chain variableregion having an amino acid sequence set forth as SEQ ID NO: 93, 94, 95,96, 97, 100, 102, or 103.

Provided herein is a method of inhibiting SMAD 1/5/8 phosphorylation infibroblasts comprising contacting cells with an antibody, orantigen-binding fragment thereof, wherein the antibody, orantigen-binding fragment thereof, comprises a heavy chain variableregion having an amino acid sequence set forth as SEQ ID NO: 89 and alight chain variable region having an amino acid sequence set forth asSEQ ID NO: 93, wherein SMAD 1/5/8 phosphorylation is inhibited.

Provided herein is a method of inhibiting SMAD 1/5/8 phosphorylation infibroblasts comprising contacting cells with an antibody, orantigen-binding fragment thereof, wherein the antibody, orantigen-binding fragment thereof, comprises a heavy chain variableregion having an amino acid sequence set forth as SEQ ID NO: 89 and alight chain variable region having an amino acid sequence set forth asSEQ ID NO: 93, wherein said heavy chain variable region furthercomprises one or more modifications selected from the group consistingof a substitution of glycine (G) by alanine (A) or serine (S) atposition 49; a substitution of alanine (A) by isoleucine (I) at position51; a substitution of lysine (K) by arginine (R) or asparagine (Q) atposition 52b; a substitution of leucine (L) by valine (V) at position 78utilizing the Kabat numbering system; and the light chain variableregion further comprises one or more modifications selected from thegroup consisting of a substitution of methionine (M) by leucine (L) atposition 4; a substitution of alanine (A) by valine (V) at position 19;a substitution of threonine (T) by serine (S) at position 22; asubstitution of alanine (A) by isoleucine (I) at position 48; and asubstitution of threonine (T) by serine (S) at position 51 utilizing theKabat numbering system, wherein SMAD 1/5/8 phosphorylation is inhibited.

In such methods, an antibody, or antigen-binding fragment thereof, cancomprise antibody, or antigen-binding fragment thereof, comprises aheavy chain variable region having an amino acid sequence set forth asSEQ ID NO: 88, 89, 90, 91 or 92; and a light chain variable regionhaving an amino acid sequence set forth as SEQ ID NO: 93, 94, 95, 96,97, 100, 102, or 103.

In any of such methods, the antibody can block binding of BMP9 toendoglin.

In one aspect, the humanized and deimmunized antibodies andantigen-binding fragments described herein can be any isotype. Alsoencompassed herein are AVIMERs, diabodies, and heavy chain dimers(including camelids and shark heavy chain constructs).

The terms “antigen-binding portion of an antibody,” “antigen-bindingfragment,” “antigen-binding domain,” “antibody fragment” or a“functional fragment of an antibody” are used interchangeably herein torefer to one or more fragments of an antibody that retain the ability tospecifically bind to an antigen. Non-limiting examples of antibodyfragments included within such terms include, but are not limited to,(i) a Fab fragment, a monovalent fragment consisting of the V_(L),V_(H), C_(L) and C_(H)1 domains; (ii) a F(ab′)₂ fragment, a bivalentfragment containing two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fd fragment consisting of the V_(H) and C_(H)1domains; (iv) a Fv fragment containing the V_(L) and V_(H) domains of asingle arm of an antibody, (v) a dAb fragment (Ward et al., (1989)Nature 341:544 546), which containing a V_(H) domain; and (vi) anisolated CDR. Additionally included in this definition are “one-half”antibodies comprising a single heavy chain and a single light chain thatretain binding functions. Other forms of single chain antibodies, suchas diabodies are also encompassed herein.

An antigen-binding fragment can be any of those described hereinincluding, but not limited to, a Fab fragment, a Fab′, a F(ab′)₂fragment, an Fv fragment (including non-covalently and covalently linkedFv fragments), an scFv fragment, a single chain binding polypeptide, anFd fragment, an Fv fragment or a dAb fragment. In one non-limitingembodiment, the antigen-binding fragment is a scFv which can,optionally, be further fused to a human Fc portion of an antibody.

In one aspect, the humanized and deimmunized antibodies andantigen-binding fragments described herein can be modified. For example,in one embodiment, the compound can be modified to alter apharmacokinetic property of the compound such as, for example, in vivostability, solubility, bioavailability or half-life. Such modificationsinclude, but are not limited to, PEGylation and/or glycosylation.

The humanized and deimmunized antibodies and antigen-binding fragmentsdescribed herein can be formulated for rapid or extended delivery usingconventional means. In one non-limiting embodiment, rapid delivery is,for example, by intravenous injection. In another non-limitingembodiment, extended delivery is, for example, by subcutaneousdeposition.

Compositions can be administered locally, regionally or systemically,such as, for example, administration by subcutaneous, subcutaneous,intravitreal, intradermal, intravenous, intra-arterial, intraperitonealor intramuscular injection.

Provided herein are compositions of the humanized and deimmunizedantibodies and antigen-binding fragments described herein and anacceptable carrier or excipient.

Antibodies and antigen-binding fragments thereof as described herein canbe used to treat various fibrosis and symptoms associated with fibrosis.Additionally, these humanized and deimmunized antibodies andantigen-binding fragments thereof described herein can be used in theformulation of a medicament for the prophylaxis, treatment, inhibition,or amelioration of fibrosis and symptoms associated with fibrosis.Fibrosis in some instances is liver fibrosis. In other instances,fibrosis is kidney fibrosis. Fibrosis in yet other instances ispulmonary fibrosis. Fibrosis in yet other instances is cardiac fibrosis.

Provided herein is a method of treating, inhibiting or amelioratingfibrosis in a subject by administering a composition provided herein toa patient. Fibrosis includes, but is not limited to, liver, kidney,lung, heart, skin, intestines, eye (ocular), joints, etc. In oneembodiment, fibrosis is liver fibrosis. In another embodiment, fibrosisis kidney fibrosis. In another embodiment, fibrosis is skin fibrosis. Inanother embodiment, fibrosis is intestinal fibrosis. In anotherembodiment, fibrosis is ocular fibrosis. In another embodiment, fibrosisis fibrosis of the joints.

The method can further include one or more supplemental treatments offibrosis. An anti-fibrotic agent can be administered prior to,concomitant with, or subsequent to, administration of the pharmaceuticalcomposition containing a humanized and deimmunized anti-endoglinantibody. If an anti-fibrotic agent is administered on the same day asthe pharmaceutical composition, administration can be concomitant orsequential. An anti-fibrotic agent can be administered at the same site,or at a different site than administration of the pharmaceuticalcomposition containing a humanized and deimmunized anti-endoglinantibody.

The invention further provides a composition comprising an endoglinbinding antibody or binding fragment thereof and a pharmaceuticallyacceptable excipient for use in treating fibrosis, wherein the antibodyor the antigen binding fragment thereof comprises a heavy chain variableregion and a light chain variable region wherein: the heavy chainvariable region comprises an amino acid sequence as set forth in SEQ IDNO: 89 optionally comprising one or more modifications selected from thegroup consisting of a substitution of glycine (G) by alanine (A) orserine (S) at position 49; a substitution of alanine (A) by isoleucine(I) at position 51; a substitution of lysine (K) by arginine (R) orasparagine (Q) at position 52b; a substitution of leucine (L) by valine(V) at position 78 utilising the Kabat number system; and wherein thelight chain variable region comprises an amino acid sequence as setforth in SEQ ID NO: 89 optionally comprising one or more modificationsselected from the group consisting of a substitution of methionine (M)by leucine (L) at position 4; a substitution of alanine (A) by valine(V) at position 19; a substitution of threonine (T) by serine (S) atposition 22; a substitution of alanine (A) by isoleucine (I) at position48; and a substitution of threonine (T) by serine(S) at position 51utilising the Kabat number system; and a pharmaceutically acceptableexcipient, whereby fibrosis is treated or inhibited.

The said antibody or fragment may comprise a heavy chain variable regionhaving an amino acid sequence as set forth in SEQ ID NO: 89.

The said antibody or fragment may comprise a light chain variable regionhaving an amino acid sequence as set forth in SEQ ID NO: 93.

The said antibody or fragment may comprise a heavy chain variable regionhaving an amino acid sequence as set forth in SEQ ID NO: 89 as modifiedas set forth above.

The said antibody or fragment may comprise a light chain variable regionhaving an amino acid sequence as set forth in SEQ ID NO: 93 as modifiedas set forth above.

In one preferred aspect, the said antibodies or fragments are for use inthe treatment of liver fibrosis.

In another preferred aspect, the said antibodies or fragments are foruse in the treatment of kidney fibrosis.

In another preferred aspect, the said antibodies or fragments are foruse in the treatment of skin fibrosis.

In another preferred aspect, the said antibodies or fragments are foruse in the treatment of intestinal fibrosis.

In another preferred aspect, the said antibodies or fragments are foruse in the treatment of ocular fibrosis.

In another preferred aspect, the said antibodies or fragments are foruse in the treatment of fibrosis of joints.

The antibodies and antigen binding fragments thereof may be humanizedand deimmunized and the preparation of antibodies having thesecharacteristics has been described in U.S. Pat. No. 8,221,753.

The antibodies may be humanized to contain an IgG1, an IgG2, an IgG3 oran IgG4 isotype. In one embodiment, an antibody may be humanized tocontain an IgG1 isotype. In one embodiment, an antibody may be humanizedto contain an IgG2 isotype. In one embodiment, an antibody may behumanized to contain an IgG3 isotype. In one embodiment, an antibody maybe humanized to contain an IgG4 isotype.

In the methods provided herein, the subject can be a human or anon-human subject. Compositions and the anti-fibrotic agent ortreatments provided herein can be administered once or multiple timesdepending on the health of the patient, the progression of the diseaseor condition, and the efficacy of the treatment. Adjustments to therapyand treatments can be made throughout the course of treatment.

Provided herein is a method of monitoring the efficacy of one or more ofany of the methods provided herein to determine whether treatment shouldbe maintained, increased, or decreased.

One embodiment of the present application contemplates the use of any ofthe compositions of the present invention to formulate a medicament fortreating, inhibiting, or ameliorating fibrosis. Medicaments can beformulated based on the physical characteristics of the subject needingtreatment, and can be formulated in single or multiple formulationsbased on the site of fibrosis. Medicaments of the present invention canbe packaged in a suitable pharmaceutical package with appropriate labelsfor the distribution to hospitals and clinics wherein the label is forthe indication of treating a disorder as described herein in a subject.Medicaments can be packaged as a single or multiple units. Instructionsfor the dosage and administration of the pharmaceutical compositions ofthe present invention can be included with the pharmaceutical packages.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference in its entiretyunless otherwise specifically noted.

U.S. Pat. No. 8,221,753, issued Jul. 17, 2012, is hereby incorporated byreference is its entirety.

International Application No. PCT/US2013/058265, filed Sep. 5, 2013,published as WO 2014/039682, is hereby incorporated by reference is itsentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 30, 2016, isnamed 35882-718.501_SL.txt and is 74,853 bytes in size.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 provides a humanized O2-Vκ1-39 variable (V_(L)) light chainhaving the monoclonal murine chimeric TRC105 V_(L) CDRs (underlined)grafted between the framework regions (FRs) 1-3 of the human sequenceO2-Vκ1-39 and a framework region 4 from the human Jκ4 sequence (SEQ IDNO: 4) (all in bold). Variations that can be made to the human FRs areindicated at positions 1, 3, 4, 5, 36, 46, 47, 60, 70, 71, 100, and 106of the sequence (sequence disclosed at SEQ ID NO: 86) utilizing theKabat numbering system (shown in italics beneath the humanizedsequence).

FIG. 2 provides a humanized VH3-15 variable (V_(H)) heavy chain havingthe monoclonal murine monoclonal murine chimeric TRC105 V_(H) CDRs(underlined) grafted between the framework regions (FRs) 1-3 of thehuman sequence VH3-15 and a framework region 4 from the human JH4sequence (SEQ ID NO: 42) (all in bold). One or more variations that canbe made to the human FRs are indicated at positions 49, 76, 77, 78, 82a,89, 94, 108, 109, and 113 of the sequence (sequence disclosed at SEQ IDNO: 87) utilizing the Kabat numbering system (shown in italics beneaththe humanized sequence).

FIGS. 3A-3B provide an amino acid sequence alignment of exemplary mouseand humanized VK chains (FIG. 3A; SEQ ID NOS 1-5, respectively, in orderof appearance) and V_(H) chains (FIG. 3B; SEQ ID NOS 39-43,respectively, in order of appearance) produced according the inventiondescribed herein.

FIGS. 4A-4B provide an amino acid sequence alignment of exemplary mouseand super-humanized VK chains (FIG. 4A; SEQ ID NOS 1 and 69-72,respectively, in order of appearance) and V_(H) chains (FIG. 4B; SEQ IDNOS 39 and 73-75, respectively, in order of appearance) producedaccording the invention described herein.

FIG. 5 provides an amino acid sequence alignment and comparison ofexemplary mouse and humanized and super-humanized VK chains (SEQ ID NOS1, 3 and 70, respectively, in order of appearance) and V_(H) chains (SEQID NOS 39, 41 and 74, respectively, in order of appearance) producedaccording the invention described herein.

FIGS. 6A-6F. Anti-CD105 competition ELISA with humanized andhumanized/deimmunized antibodies. Varying concentrations of eachantibody were mixed with a fixed concentration of biotinylated referenceanti-CD105 antibody (6.25 ng/ml) and bound to CD105 (100 ng/ml) capturedon a Nunc MaxiSorp plate. Binding was detected via streptavidin-HRP andTMB substrate. Absorbance (OD) at 450 nm was measured on a plate readerand this was plotted against the test antibody concentration. FIG. 6Aprovides the results from the chimeric control compared to VK1VH1,VK1VH2, VK2VH1 and VK2VH2. FIG. 6B provides the results from thechimeric control compared to VK1VH1, VK2AVH1A, VK2SAVH1Q, VK2SAVH1R andVK2SAVH1S. FIG. 6C provides the results from the chimeric controlcompared to VK2AAVH1A2, VK2ASVH1A2, VK2ASVH1Q, VK2SSVH1A and VK2SSVH1Q.FIG. 6D provides the results from the chimeric control compared toVK2VH1, VK2AAVH1A, VK2AAVH1Q, VK2AAVH1R and VK2AAVH1S. FIG. 6E providesthe results from the chimeric control compared to VK2VH1, VK2ASVH1A,VK2ASVH1R, VK2ASVH1S, and VK2SAVH1A2. FIG. 6F provides the results fromthe chimeric control compared to VK2VH1, VK2SSVH1Q, VK2SSVH1A2,VK2SSVH1R and VK2SSVH1S.

FIG. 7 illustrates the lead humanized deimmunized heavy chain variableregion with CDRs in bold and underlined (sequence disclosed at SEQ IDNO: 89). Variations that can be made are indicated at the identifiedpositions of the sequence utilizing the Kabat numbering system (sequencedisclosed at SEQ ID NO: 116) (shown in italics beneath the humanizedsequence). Variations may be made as a single mutation or as more thanone mutation, and variations can be made with mutations in anycombination.

FIG. 8 illustrates the lead humanized deimmunized light chain variableregion with CDRs in bold and underlined (sequence disclosed at SEQ IDNO: 93). Variations that can be made are indicated at the identifiedpositions of the sequence utilizing the Kabat numbering system (sequencedisclosed at SEQ ID NO: 117) (shown in italics beneath the humanizedsequence). Variations may be made as a single mutation or as more thanone mutation, and variations can be made with mutations in anycombination.

FIG. 9 illustrates binding illustrates binding of BMP 9 to the substratebinding site of endoglin (left panel) and illustrates binding of ahumanized and deimmunized anti-endoglin antibody binding to endoglin andpreventing binding of BMP9 (right panel).

FIG. 10 illustrates body weight changes of mice in an in vivo CarbonTetrachloride-induced Liver Fibrosis Model in disease-control animals,in animals treated with an isotype-matched antibody, and in animalstreated with M1043 Antibody.

FIGS. 11A-B illustrate body weight and liver weight. FIG. 11A provides agraph of body weight for the three groups. FIG. 11B provides a graph ofliver weight for the three groups.

FIGS. 12A-F are representative photomicrographs of Sirius red-stainedliver sections. FIG. 12A is a ×100 picture of a liver section fromdisease-control animals. FIG. 12B is a ×200 picture of a liver sectionfrom disease-control animals. FIG. 12C is a ×100 picture of a liversection of animals treated with an isotype-matched antibody. FIG. 12D isa ×200 picture of a liver section of animals treated with anisotype-matched antibody. FIG. 12E is a ×100 picture of a liver sectionof animals treated with M1043 Antibody. FIG. 12F is a ×200 picture of aliver section of animals treated with M1043 Antibody.

FIG. 13 is a graph illustrating the liver fibrotic area (% Siriusred-positive area) in disease-control animals, in animals treated withan isotype-matched antibody, and in animals treated with M1043 Antibody.

FIGS. 14A-E. Results of the bleomycin induced pulmonary fibrosis model.FIG. 14A. Lung hydroxyproline was quantified by a hydrolysis method.FIG. 14B. Histological analysis of lung sections was conducted bypractitioners who are not aware of the Group identity. Masson'sTrichrome staining is conducted and an Ashcroft Score estimate wasdetermined. FIG. 14C. There were no significant differences in bodyweight between the five treatment groups by Bonferroni MultipleComparison Testing (p>0.05 comparing the isotype control antibody groupversus each other group). FIG. 14D. There were no significantdifferences in survival between the five treatment groups by BonferroniMultiple Comparison Testing (p>0.05 comparing the isotype controlantibody group versus each other group). FIG. 14E. Ashcroft scores.

FIG. 15: ELISA was established to determine the binding of M1043antibody to mouse endoglin was determined by ELISA.

FIG. 16: Competition ELISA of biotinylated M1043 with BMP9 binding toendoglin. A dilution series of BMP9 was competed against a fixedconcentration of biotinylated M1043 for binding to endoglin. Rat IgG1was used as a negative control. Bound biotinylated M1043 antibody wasdetected with streptavidin-HRP conjugate and TMB substrate.

FIG. 17. The binding of biotinylated TRC205 antibody to human CD105 wasdetermined by ELISA.

FIG. 18. The ability of human BMP9 to compete with the TRC205 antibodyfor binding to human CD105 was determined by competition ELISA.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that this application is not limited toparticular formulations or process parameters, as these may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to be limiting. Further, it is understood that a number ofmethods and materials similar or equivalent to those described hereincan be used in the practice of the present inventions.

In accordance with the present application, there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook et al., “Molecular Cloning:A Laboratory Manual” (1989); “Current Protocols in Molecular Biology”Volumes I-III [Ausubel, R. M., ed. (1994)]; “Cell Biology: A LaboratoryHandbook” Volumes I-III [J. E. Celis, ed. (1994))]; “Current Protocolsin Immunology” Volumes I-III [Coligan, J. E., ed. (1994)];“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “TranscriptionAnd Translation” [B. D. Hames & S. J. Higgins, eds. (1984)]; “AnimalCell Culture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells AndEnzymes” [IRL Press, (1986)]; B. Perbal, “A Practical Guide To MolecularCloning” (1984), each of which is specifically incorporated herein byreference in its entirety.

I. Anti-Endoglin Antibodies

Provided herein are humanized antibodies, and antigen-binding fragmentsthereof that bind endoglin. Humanized and deimmunized antibodiesdescribed herein exhibit reduced immunogenicity while maintaining and/orimproving their specificity. These humanized and deimmunized endoglinantibodies are useful for the treatment, inhibition or amelioration offibrosis as well as for purification and detection of endoglin, in vitroassays, and treatment of subjects suffering from fibrosis.

The present inventors have identified that a neutralizing antibody orantigen-binding fragment thereof that specifically binds to endoglin canbind to endoglin and inhibit binding of BMP9 to a receptor in the TGFbeta family.

The inventors have also newly described a method of inhibiting BMPsignaling in fibroblasts by virtue of the antibody or fragment thereofblocking binding of BMP9 to endoglin.

Also described herein is a method of inhibiting fibrosis byadministering an antibody or antigen-binding fragment thereof thatspecifically binds to an endoglin receptor and inhibits Smad 1/5/8signaling.

A. Antibody Terminology

As used herein, the term “antibody” refers to an immunoglobulin (Ig)whether natural or partly or wholly synthetically produced. The termalso covers any polypeptide or protein having a binding domain which is,or is homologous to, an antigen-binding domain. The term furtherincludes “antigen-binding fragments” and other interchangeable terms forsimilar binding fragments such as described below. Complementaritydetermining region (CDR) grafted antibodies and other humanizedantibodies (including CDR modifications and framework regionmodifications) are also contemplated by this term.

Hereinafter, a reference to the terms “antibody” or “antibodies” are tobe considered inclusive of any of the antigen-binding fragmentsdescribed herein and the terms are to be interchangeable whereapplicable. In addition to their use for purification of endoglin, theseantibodies are useful for purification, detection and diagnosticpurposes as well as therapeutic purposes. The antibodies provided hereincan be used for the formulation of medicaments for the treatment offibrosis.

Native antibodies and native immunoglobulins are usuallyheterotetrameric glycoproteins of about 150,000 Daltons, composed of twoidentical light (L) chains and two identical heavy (H) chains. Eachlight chain is typically linked to a heavy chain by one covalentdisulfide bond, while the number of disulfide linkages varies among theheavy chains of different immunoglobulin isotypes. Each heavy and lightchain also has regularly spaced intrachain disulfide bridges. Each heavychain has at one end a variable domain (“V_(H)”) followed by a number ofconstant domains (“C_(H)”). Each light chain has a variable domain atone end (“V_(L)”) and a constant domain (“C_(L)”) at its other end; theconstant domain of the light chain is aligned with the first constantdomain of the heavy chain, and the light-chain variable domain isaligned with the variable domain of the heavy chain. Particular aminoacid residues are believed to form an interface between the light- andheavy-chain variable domains.

The terms “synthetic polynucleotide,” “synthetic gene” or “syntheticpolypeptide,” as used herein, mean that the corresponding polynucleotidesequence or portion thereof, or amino acid sequence or portion thereof,is derived, from a sequence that has been designed, or synthesized denovo, or modified, compared to an equivalent naturally-occurringsequence. Synthetic polynucleotides (antibodies or antigen bindingfragments) or synthetic genes can be prepared by methods known in theart, including but not limited to, the chemical synthesis of nucleicacid or amino acid sequences. Synthetic genes are typically differentfrom naturally-occurring genes, either at the amino acid, orpolynucleotide level, (or both) and are typically located within thecontext of synthetic expression control sequences. For example,synthetic gene sequences can include amino acid, or polynucleotide,sequences that have been changed, for example, by the replacement,deletion, or addition, of one or more, amino acids, or nucleotides,thereby providing an antibody amino acid sequence, or a polynucleotidecoding sequence that is different from the source sequence. Syntheticgene polynucleotide sequences, may not necessarily encode proteins withdifferent amino acids, compared to the natural gene; for example, theycan also encompass synthetic polynucleotide sequences that incorporatedifferent codons but which encode the same amino acid (i.e., thenucleotide changes represent silent mutations at the amino acid level).

With respect to antibodies, the term “variable domain” refers to thevariable domains of antibodies that are used in the binding andspecificity of each particular antibody for its particular antigen.However, the variability is not evenly distributed throughout thevariable domains of antibodies. Rather, it is concentrated in threesegments called hypervariable regions (also known as CDRs) in both thelight chain and the heavy chain variable domains. More highly conservedportions of variable domains are called the “framework regions” or“FRs.” The variable domains of unmodified heavy and light chains eachcontain four FRs (FR1, FR2, FR3 and FR4), largely adopting a β-sheetconfiguration interspersed with three CDRs which form loops connectingand, in some cases, part of the β-sheet structure. The CDRs in eachchain are held together in close proximity by the FRs and, with the CDRsfrom the other chain, contribute to the formation of the antigen-bindingsite of antibodies (see Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991), pages 647-669).

The terms “hypervariable region” and “CDR” when used herein, refer tothe amino acid residues of an antibody which are responsible forantigen-binding. The CDRs comprise amino acid residues from threesequence regions which bind in a complementary manner to an antigen andare known as CDR1, CDR2, and CDR3 for each of the V_(H) and V_(L)chains. In the light chain variable domain, the CDRs typicallycorrespond to approximately residues 24-34 (CDRL1), 50-56 (CDRL2) and89-97 (CDRL3), and in the heavy chain variable domain the CDRs typicallycorrespond to approximately residues 31-35 (CDRH1), 50-65 (CDRH2) and95-102 (CDRH3) according to Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)). It is understood that theCDRs of different antibodies may contain insertions, thus the amino acidnumbering may differ. The Kabat numbering system accounts for suchinsertions with a numbering scheme that utilizes letters attached tospecific residues (e.g., 27A, 27B, 27C, 27D, 27E, and 27F of CDRL1 inthe light chain) to reflect any insertions in the numberings betweendifferent antibodies. Alternatively, in the light chain variable domain,the CDRs typically correspond to approximately residues 26-32 (CDRL1),50-52 (CDRL2) and 91-96 (CDRL3), and in the heavy chain variable domain,the CDRs typically correspond to approximately residues 26-32 (CDRH1),53-55 (CDRH2) and 96-101 (CDRH3) according to Chothia and Lesk, J. Mol.Biol., 196: 901-917 (1987)).

As used herein, “framework region” or “FR” refers to framework aminoacid residues that form a part of the antigen binding pocket or groove.In some embodiments, the framework residues form a loop that is a partof the antigen binding pocket or groove and the amino acids residues inthe loop may or may not contact the antigen. Framework regions generallycomprise the regions between the CDRs. In the light chain variabledomain, the FRs typically correspond to approximately residues 0-23(FRL1), 35-49 (FRL2), 57-88 (FRL3), and 98-109 and in the heavy chainvariable domain the FRs typically correspond to approximately residues0-30 (FRH1), 36-49 (FRH2), 66-94 (FRH3), and 103-133 according to Kabatet al., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991)). Asdiscussed above with the Kabat numbering for the light chain, the heavychain too accounts for insertions in a similar manner (e.g., 35A, 35B ofCDRH1 in the heavy chain). Alternatively, in the light chain variabledomain, the FRs typically correspond to approximately residues 0-25(FRL1), 33-49 (FRL2) 53-90 (FRL3), and 97-109 (FRL4), and in the heavychain variable domain, the FRs typically correspond to approximatelyresidues 0-25 (FRH1), 33-52 (FRH2), 56-95 (FRH3), and 102-113 (FRH4)according to Chothia and Lesk, J. Mol. Biol., 196: 901-917 (1987)).

The loop amino acids of a FR can be assessed and determined byinspection of the three-dimensional structure of an antibody heavy chainand/or antibody light chain. The three-dimensional structure can beanalyzed for solvent accessible amino acid positions as such positionsare likely to form a loop and/or provide antigen contact in an antibodyvariable domain. Some of the solvent accessible positions can tolerateamino acid sequence diversity and others (e.g., structural positions)are, generally, less diversified. The three dimensional structure of theantibody variable domain can be derived from a crystal structure orprotein modeling.

Constant domains (Fc) of antibodies are not involved directly in bindingan antibody to an antigen but, rather, exhibit various effectorfunctions, such as participation of the antibody in antibody-dependentcellular toxicity via interactions with, for example, Fc receptors(FcR). Fc domains can also increase bioavailability of an antibody incirculation following administration to a patient.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these can be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Theheavy-chain constant domains (Fc) that correspond to the differentclasses of immunoglobulins are called α, δ, ϵ, γ, and μ, respectively.The subunit structures and three-dimensional configurations of differentclasses of immunoglobulins are well known. The functionalcharacteristics of the different classes of immunoglobulins are alsoknown.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa or (“κ” or “K”) and lambda or (“λ”), based on the amino acidsequences of their constant domains.

The terms “antigen-binding portion of an antibody,” “antigen-bindingfragment,” “antigen-binding domain,” “antibody fragment” or a“functional fragment of an antibody” are used interchangeably herein torefer to one or more fragments of an antibody that retain the ability tospecifically bind to an antigen. Non-limiting examples of antibodyfragments included within such terms include, but are not limited to,(i) a Fab fragment, a monovalent fragment consisting of the V_(L),V_(H), C_(L) and C_(H1) domains; (ii) a F(ab′)₂ fragment, a bivalentfragment containing two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fd fragment consisting of the V_(H) and C_(H1)domains; (iv) a Fv fragment containing the V_(L) and V_(H) domains of asingle arm of an antibody, (v) a dAb fragment (Ward et al., (1989)Nature 341:544 546), which containing a V_(H) domain; and (vi) anisolated CDR. Additionally included in this definition are “one-half”antibodies comprising a single heavy chain and a single light chain.Other forms of single chain antibodies, such as diabodies are alsoencompassed herein.

“F(ab′)₂” and “Fab′” moieties can be produced by treating an Ig with aprotease such as pepsin and papain, and include antibody fragmentsgenerated by digesting immunoglobulin near the disulfide bonds existingbetween the hinge regions in each of the two heavy chains. For example,papain cleaves IgG upstream of the disulfide bonds existing between thehinge regions in each of the two heavy chains to generate two homologousantibody fragments in which an light chain composed of V_(L) and C_(L)(light chain constant region), and a heavy chain fragment composed ofV_(H) and C_(Hγ1) (γ1) region in the constant region of the heavy chain)are connected at their C terminal regions through a disulfide bond. Eachof these two homologous antibody fragments is called Fab′. Pepsin alsocleaves IgG downstream of the disulfide bonds existing between the hingeregions in each of the two heavy chains to generate an antibody fragmentslightly larger than the fragment in which the two above-mentioned Fab′are connected at the hinge region. This antibody fragment is calledF(ab′)₂.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (C_(H)1) of the heavy chain. Fab′fragments differ from Fab fragments by the addition of a few residues atthe carboxyl terminus of the heavy chain C_(H)1 domain including one ormore cysteine(s) from the antibody hinge region. Fab′-SH is thedesignation herein for Fab′ in which the cysteine residue(s) of theconstant domains bear a free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

“Fv” refers to an antibody fragment which contains a completeantigen-recognition and antigen-binding site. This region consists of adimer of one heavy chain and one light chain variable domain in tight,non-covalent or covalent association (disulfide linked Fv's have beendescribed in the art, Reiter et al. (1996) Nature Biotechnology14:1239-1245). It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, a combination of one ormore of the CDRs from each of the V_(H) and V_(L) chains conferantigen-binding specificity to the antibody. For example, it would beunderstood that, for example, the CDRH3 and CDRL3 could be sufficient toconfer antigen-binding specificity to an antibody when transferred toV_(H) and V_(L) chains of a recipient antibody or antigen-bindingfragment thereof and this combination of CDRs can be tested for binding,affinity, etc. using any of the techniques described herein. Even asingle variable domain (or half of an Fv comprising only three CDRsspecific for an antigen) has the ability to recognize and bind antigen,although likely at a lower affinity than when combined with a secondvariable domain. Furthermore, although the two domains of a Fv fragment(V_(L) and V_(H)), are coded for by separate genes, they can be joinedusing recombinant methods by a synthetic linker that enables them to bemade as a single protein chain in which the V_(L) and V_(H) regions pairto form monovalent molecules (known as single chain Fv (scFv); Bird etal. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad.Sci. USA 85:5879-5883; and Osbourn et al. (1998) Nat. Biotechnol.16:778). Such scFvs are also intended to be encompassed within the term“antigen-binding portion” of an antibody. Any V_(H) and V_(L) sequencesof specific scFv can be linked to an Fc region cDNA or genomicsequences, in order to generate expression vectors encoding complete Ig(e.g., IgG) molecules or other isotypes. V_(H) and V_(L) can also beused in the generation of Fab, Fv or other fragments of Igs using eitherprotein chemistry or recombinant DNA technology.

“Single-chain Fv” or “sFv” antibody fragments comprise the V_(H) andV_(L) domains of an antibody, wherein these domains are present in asingle polypeptide chain. In some embodiments, the Fv polypeptidefurther comprises a polypeptide linker between the V_(H) and V_(L)domains which enables the sFv to form the desired structure for antigenbinding. For a review of sFvs, see, e.g., Pluckthun in The Pharmacologyof Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds.Springer-Verlag, New York, pp. 269-315 (1994).

The term “AVIMER™” refers to a class of therapeutic proteins of humanorigin, which are unrelated to antibodies and antibody fragments, andare composed of several modular and reusable binding domains, referredto as A-domains (also referred to as class A module, complement typerepeat, or LDL-receptor class A domain). They were developed from humanextracellular receptor domains by in vitro exon shuffling and phagedisplay (Silverman et al., 2005, Nat. Biotechnol. 23:1493-1494;Silverman et al., 2006, Nat. Biotechnol. 24:220). The resulting proteinscan contain multiple independent binding domains that can exhibitimproved affinity (in some cases, sub-nanomolar) and specificitycompared with single-epitope binding proteins. See, for example, U.S.Patent Application Publ. Nos. 2005/0221384, 2005/0164301, 2005/0053973and 2005/0089932, 2005/0048512, and 2004/0175756, each of which ishereby incorporated by reference herein in its entirety.

Each of the known 217 human A-domains comprises ˜35 amino acids (˜4kDa); and these domains are separated by linkers that average five aminoacids in length. Native A-domains fold quickly and efficiently to auniform, stable structure mediated primarily by calcium binding anddisulfide formation. A conserved scaffold motif of only 12 amino acidsis required for this common structure. The end result is a singleprotein chain containing multiple domains, each of which represents aseparate function. Each domain of the proteins binds independently andthe energetic contributions of each domain are additive. These proteinswere called “AVIMERs™” from avidity multimers.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (V_(H)) connected to a light chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA 90:6444 6448 (1993).

Antigen-binding polypeptides also include heavy chain dimers such as,for example, antibodies from camelids and sharks. Camelid and sharkantibodies comprise a homodimeric pair of two chains of V-like andC-like domains (neither has a light chain). Since the V_(H) region of aheavy chain dimer IgG in a camelid does not have to make hydrophobicinteractions with a light chain, the region in the heavy chain thatnormally contacts a light chain is changed to hydrophilic amino acidresidues in a camelid. V_(H) domains of heavy-chain dimer IgGs arecalled V_(HH) domains. Shark Ig-NARs comprise a homodimer of onevariable domain (termed a V-NAR domain) and five C-like constant domains(C-NAR domains). In camelids, the diversity of antibody repertoire isdetermined by the CDRs 1, 2, and 3 in the V_(H) or V_(HH) regions. TheCDR3 in the camel V_(HH) region is characterized by its relatively longlength, averaging 16 amino acids (Muyldermans et al., 1994, ProteinEngineering 7(9): 1129). This is in contrast to CDR3 regions ofantibodies of many other species. For example, the CDR3 of mouse V_(H)has an average of 9 amino acids. Libraries of camelid-derived antibodyvariable regions, which maintain the in vivo diversity of the variableregions of a camelid, can be made by, for example, the methods disclosedin U.S. Patent Application Ser. No. 20050037421.

“Humanized” forms of non-human (e.g., murine) antibodies includechimeric antibodies which contain minimal sequence derived from anon-human Ig. For the most part, humanized antibodies are human Igs(recipient antibody) in which one or more of the CDRs of the recipientare replaced by CDRs from a non-human species antibody (donor antibody)such as mouse, rat, rabbit or non-human primate having the desiredspecificity, affinity and binding function. In some instances, one ormore FR amino acid residues of the human Ig are replaced bycorresponding non-human amino acid residues. Furthermore, humanizedantibodies can contain residues which are not found in the recipientantibody or in the donor antibody. These modifications can be made torefine antibody performance, if needed. A humanized antibody cancomprise substantially all of at least one and, in some cases two,variable domains, in which all or substantially all of the hypervariableregions correspond to those of a non-human immunoglobulin and all, orsubstantially all, of the FRs are those of a human immunoglobulinsequence. The humanized antibody optionally can also include at least aportion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin. For details, see Jones et al., Nature 321: 522-525(1986); Reichmann et al., Nature 332: 323-329 (1988); and Presta, Curr.Op. Struct. Biol. 2: 593-596 (1992).

A humanized antibody also includes antibodies in which part, or all ofthe CDRs of the heavy and light chain are derived from a non-humanmonoclonal antibody, substantially all the remaining portions of thevariable regions are derived from human variable region (both heavy andlight chain), and the constant regions are derived from a human constantregion. In one embodiment, the CDR1, CDR2 and CDR3 regions of the heavyand light chains are derived from a non-human antibody. In yet anotherembodiment, at least one CDR (e.g., a CDR3) of the heavy and lightchains is derived from a non-human antibody. Various combinations ofCDR1, CDR2, and CDR3 can be derived from a non-human antibody and arecontemplated herein. In one non-limiting example, one or more of theCDR1, CDR2 and CDR3 regions of each of the heavy and light chains arederived from a murine chimeric monoclonal antibody clone TRC105.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations, which can includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, monoclonal antibodiescan be made by the hybridoma method first described by Kohler et al.,Nature 256:495 (1975), or can be made by recombinant DNA methods (see,e.g., U.S. Pat. No. 4,816,567). In certain embodiments, the monoclonalantibodies can be isolated from phage antibody libraries using thetechniques described in Clackson et al., Nature 352:624-628 (1991) andMarks et al., J. Mol. Biol. 222:581-597 (1991), for example.

Antibodies can be isolated and purified from the culture supernatant orascites mentioned above by saturated ammonium sulfate precipitation,euglobulin precipitation method, caproic acid method, caprylic acidmethod, ion exchange chromatography (DEAE or DE52), or affinitychromatography using anti-Ig column or a protein A, G or L column suchas described in more detail below.

Exemplary antibodies for use in the compositions and methods describedherein are intact immunoglobulin molecules, such as, for example, ahumanized antibody or those portions of a humanized Ig molecule thatcontain the antigen binding site (i.e., paratope) or a single heavychain and a single light chain, including those portions known in theart as Fab, Fab′, F(ab)′, F(ab′)₂, Fd, scFv, a variable heavy domain, avariable light domain, a variable NAR domain, bi-specific scFv, abi-specific Fab₂, a tri-specific Fab₃ and a single chain bindingpolypeptides and others also referred to as antigen-binding fragments.When constructing an immunoglobulin molecule or fragments thereof,variable regions or portions thereof may be fused to, connected to, orotherwise joined to one or more constant regions or portions thereof toproduce any of the antibodies or fragments thereof described herein.This may be accomplished in a variety of ways known in the art,including but not limited to, molecular cloning techniques or directsynthesis of the nucleic acids encoding the molecules. Exemplarynon-limiting methods of constructing these molecules can also be foundin the examples described herein.

In one exemplary embodiment, the application contemplates a single chainbinding polypeptide having a heavy chain variable region, and/or a lightchain variable region which binds endoglin and has, optionally, animmunoglobulin Fc region. In one exemplary embodiment, the applicationcontemplates a single chain binding polypeptide having a heavy chainvariable region, and/or a light chain variable region which bindsendoglin and has, optionally, an immunoglobulin Fc region. Such amolecule is a single chain variable fragment optionally having effectorfunction or increased half-life through the presence of theimmunoglobulin Fc region. Methods of preparing single chain bindingpolypeptides are known in the art (e.g., U.S. Patent Application No.2005/0238646).

The terms “germline gene segments” or “germline sequences” refer to thegenes from the germline (the haploid gametes and those diploid cellsfrom which they are formed). The germline DNA contains multiple genesegments that encode a single Ig heavy or light chain. These genesegments are carried in the germ cells but cannot be transcribed andtranslated into heavy and light chains until they are arranged intofunctional genes. During B-cell differentiation in the bone marrow,these gene segments are randomly shuffled by a dynamic genetic systemcapable of generating more than 10⁸ specificities. Most of these genesegments are published and collected by the germline database.

As used herein, “immunoreactive” refers to binding agents, antibodies orfragments thereof that are specific to a sequence of amino acid residues(“binding site” or “epitope”), yet if are cross-reactive to otherpeptides/proteins, are not toxic at the levels at which they areformulated for administration to human use. The term “binding” refers toa direct association between two molecules, due to, for example,covalent, electrostatic, hydrophobic, and ionic and/or hydrogen-bondinteractions under physiological conditions, and including interactionssuch as salt bridges and water bridges and any other conventionalbinding means. The term “preferentially binds” means that the bindingagent binds to the binding site with greater affinity than it bindsunrelated amino acid sequences. Preferably such affinity is at least1-fold greater, at least 2-fold greater, at least 3-fold greater, atleast 4-fold greater, at least 5-fold greater, at least 6-fold greater,at least 7-fold greater, at least 8-fold greater, at least 9-foldgreater, 10-fold greater, at least 20-fold greater, at least 30-foldgreater, at least 40-fold greater, at least 50-fold greater, at least60-fold greater, at least 70-fold greater, at least 80-fold greater, atleast 90-fold greater, at least 100-fold greater, or at least 1000-foldgreater than the affinity of the binding agent for unrelated amino acidsequences. The terms “immunoreactive” and “preferentially binds” areused interchangeably herein.

As used herein, the term “affinity” refers to the equilibrium constantfor the reversible binding of two agents and is expressed as K_(D).Affinity of a binding protein to a ligand such as affinity of anantibody for an epitope can be, for example, from about 100 nanomolar(nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), orfrom about 100 nM to about 1 femtomolar (fM). As used herein, the term“avidity” refers to the resistance of a complex of two or more agents todissociation after dilution. Apparent affinities can be determined bymethods such as an enzyme linked immunosorbent assay (ELISA) or anyother technique familiar to one of skill in the art. Avidities can bedetermined by methods such as a Scatchard analysis or any othertechnique familiar to one of skill in the art.

“Epitope” refers to that portion of an antigen or other macromoleculecapable of forming a binding interaction with the variable regionbinding pocket of an antibody. Such binding interactions can bemanifested as an intermolecular contact with one or more amino acidresidues of one or more CDRs. Antigen binding can involve, for example,a CDR3 or a CDR3 pair or, in some cases, interactions of up to all sixCDRs of the V_(H) and V_(L) chains. An epitope can be a linear peptidesequence (i.e., “continuous”) or can be composed of noncontiguous aminoacid sequences (i.e., “conformational” or “discontinuous”). An antibodycan recognize one or more amino acid sequences; therefore an epitope candefine more than one distinct amino acid sequence. Epitopes recognizedby antibodies can be determined by peptide mapping and sequence analysistechniques well known to one of skill in the art. Binding interactionsare manifested as intermolecular contacts with one or more amino acidresidues of a CDR. TRC105 is a chimeric antibody which is the samevariable amino acid sequence as the murine antibody described as Y4-2F1or SN6j in U.S. Pat. Nos. 5,928,641; 6,200,566; 6,190,660; and7,097,836. Epitopes recognized by Y4-2F1 and SN6j, and thus TRC105, havebeen previously identified.

The term “specific” refers to a situation in which an antibody will notshow any significant binding to molecules other than the antigencontaining the epitope recognized by the antibody. The term is alsoapplicable where for example, an antigen binding domain is specific fora particular epitope which is carried by a number of antigens, in whichcase the antibody or antigen-binding fragment thereof carrying theantigen binding domain will be able to bind to the various antigenscarrying the epitope. The terms “preferentially binds” or “specificallybinds” mean that the antibodies or fragments thereof bind to an epitopewith greater affinity than it binds unrelated amino acid sequences, and,if cross-reactive to other polypeptides containing the epitope, are nottoxic at the levels at which they are formulated for administration tohuman use. In one aspect, such affinity is at least 1-fold greater, atleast 2-fold greater, at least 3-fold greater, at least 4-fold greater,at least 5-fold greater, at least 6-fold greater, at least 7-foldgreater, at least 8-fold greater, at least 9-fold greater, 10-foldgreater, at least 20-fold greater, at least 30-fold greater, at least40-fold greater, at least 50-fold greater, at least 60-fold greater, atleast 70-fold greater, at least 80-fold greater, at least 90-foldgreater, at least 100-fold greater, or at least 1000-fold greater thanthe affinity of the antibody or fragment thereof for unrelated aminoacid sequences. The terms “immunoreactive,” “binds,” “preferentiallybinds” and “specifically binds” are used interchangeably herein. Theterm “binding” refers to a direct association between two molecules, dueto, for example, covalent, electrostatic, hydrophobic, and ionic and/orhydrogen-bond interactions under physiological conditions, and includesinteractions such as salt bridges and water bridges, as well as anyother conventional means of binding.

The phrase “conservative amino acid substitution” refers to grouping ofamino acids on the basis of certain common properties. A functional wayto define common properties between individual amino acids is to analyzethe normalized frequencies of amino acid changes between correspondingproteins of homologous organisms (Schulz, G. E. and R. H. Schirmer,Principles of Protein Structure, Springer-Verlag). According to suchanalyses, groups of amino acids may be defined where amino acids withina group exchange preferentially with each other, and therefore resembleeach other most in their impact on the overall protein structure(Schulz, G. E. and R. H. Schirmer, Principles of Protein Structure,Springer-Verlag). Examples of amino acid groups defined in this mannerinclude:

(i) a charged group, consisting of Glu and Asp, Lys, Arg and His,

(ii) a positively-charged group, consisting of Lys, Arg and His,

(iii) a negatively-charged group, consisting of Glu and Asp,

(iv) an aromatic group, consisting of Phe, Tyr and Trp,

(v) a nitrogen ring group, consisting of His and Trp,

(vi) a large aliphatic non-polar group, consisting of Val, Leu and Ile,

(vii) a slightly-polar group, consisting of Met and Cys,

(viii) a small-residue group, consisting of Ser, Thr, Asp, Asn, Gly,Ala, Glu, Gln and Pro,

(ix) an aliphatic group consisting of Val, Leu, Ile, Met and Cys, and

(x) a small hydroxyl group consisting of Ser and Thr.

In addition to the groups presented above, each amino acid residue mayform its own group, and the group formed by an individual amino acid maybe referred to simply by the one and/or three letter abbreviation forthat amino acid commonly used in the art as described above.

A “conserved residue” is an amino acid that is relatively invariantacross a range of similar proteins. Often conserved residues will varyonly by being replaced with a similar amino acid, as described above for“conservative amino acid substitution.”

A “non-conservative amino acid substitution” refers to a change from oneamino acid in a group identified above into a different group.

The letter “x” or “xaa” as used in amino acid sequences herein isintended to indicate that any of the twenty standard amino acids may beplaced at this position unless specifically noted otherwise. For thepurposes of peptidomimetic design, an “x” or a “xaa” in an amino acidsequence may be replaced by a mimic of the amino acid present in thetarget sequence, or the amino acid may be replaced by a spacer ofessentially any form that does not interfere with the activity of thepeptidomimetic.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or between two nucleic acid molecules. Homology andidentity can each be determined by comparing a position in each sequencewhich may be aligned for purposes of comparison. When an equivalentposition in the compared sequences is occupied by the same base or aminoacid, then the molecules are identical at that position; when theequivalent site occupied by the same or a similar amino acid residue(e.g., similar in steric and/or electronic nature), then the moleculescan be referred to as homologous (similar) at that position. Expressionas a percentage of homology/similarity or identity refers to a functionof the number of identical or similar amino acids at positions shared bythe compared sequences. A sequence which is “unrelated” or“non-homologous” shares less than 40% identity, though preferably lessthan 25% identity with a sequence of the present invention. In comparingtwo sequences, the absence of residues (amino acids or nucleic acids) orpresence of extra residues also decreases the identity andhomology/similarity.

The term “homology” describes a mathematically based comparison ofsequence similarities which is used to identify genes or proteins withsimilar functions or motifs. The nucleic acid (nucleotide,oligonucleotide) and amino acid (protein) sequences of the presentinvention may be used as a “query sequence” to perform a search againstpublic databases to, for example, identify other family members, relatedsequences or homologs. Such searches can be performed using the NBLASTand)(BLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol.Biol. 215:403-10. BLAST nucleotide searches can be performed with theNBLAST program, score=100, word length=12 to obtain nucleotide sequenceshomologous to nucleic acid molecules of the invention. BLAST amino acidsearches can be performed with the)(BLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to protein moleculesof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al., (1997)Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and GappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and BLAST) can be used (see, world wide web sitencbi.nlm.nih.gov).

As used herein, “identity” means the percentage of identical nucleotideor amino acid residues at corresponding positions in two or moresequences when the sequences are aligned to maximize sequence matching,i.e., taking into account gaps and insertions. Identity can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., Ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,Eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., Eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988). Methods to determine identity are designed to give the largestmatch between the sequences tested. Moreover, methods to determineidentity are codified in publicly available computer programs. Computerprogram methods to determine identity between two sequences include, butare not limited to, the GCG program package (Devereux, J., et al.,Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA(Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990) andAltschul et al. Nuc. Acids Res. 25: 3389-3402 (1997)). The BLAST Xprogram is publicly available from NCBI and other sources (BLAST Manual,Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., etal., J. Mol. Biol. 215: 403-410 (1990). The well known Smith Watermanalgorithm may also be used to determine identity.

“Isolated” (used interchangeably with “substantially pure”) when appliedto polypeptides means a polypeptide or a portion thereof which, byvirtue of its origin or manipulation: (i) is present in a host cell asthe expression product of a portion of an expression vector; or (ii) islinked to a protein or other chemical moiety other than that to which itis linked in nature; or (iii) does not occur in nature, for example, aprotein that is chemically manipulated by appending, or adding at leastone hydrophobic moiety to the protein so that the protein is in a formnot found in nature. By “isolated” it is further meant a protein thatis: (i) synthesized chemically; or (ii) expressed in a host cell andpurified away from associated and contaminating proteins. The termgenerally means a polypeptide that has been separated from otherproteins and nucleic acids with which it naturally occurs. Preferably,the polypeptide is also separated from substances such as antibodies orgel matrices (polyacrylamide) which are used to purify it.

“Inducing a host immune response” means that a patient experiencesalleviation or reduction of signs or symptoms of illness, andspecifically includes, without limitation, prolongation of survival. Incertain preferred embodiments of the methods according to the invention,a CD8+ IFN-γ producing T cell is activated to induce a cytotoxic Tlymphocyte (CTL) immune response in the patient administered theantagonist. In certain embodiments of the methods according to theinvention, a CD4+ IFN-γ producing T cell is activated to induce a helperT cell immune response in the patient administered with the composition.These activated CD4+ IFN-γ producing T cells (i.e., helper T cells)provide necessary immunological help (e.g., by release of cytokines) toinduce and maintain not only CTL, but also a humoral immune responsemediated by B cells. Thus, in certain embodiments of the methodsaccording to the invention, a humoral response to the antigen isactivated in the patient administered with the composition. In oneaspect, an adjuvant may be added to the composition to increase animmune response. Adjuvants are well-known in the art.

Activation of a CD8+ and/or CD4+ T cells means causing T cells that havethe ability to produce cytokines (e.g., IFN-γ) to actually produce oneor more cytokine(s), or to increase their production of one or morecytokine(s). “Induction of CTL response” means causing potentiallycytotoxic T lymphocytes to exhibit antigen specific cytotoxicity.“Antigen specific cytotoxicity” means cytotoxicity against a cellpresenting an antigen that is associated with the antigen associatedwith the cancer that is greater than an antigen that is not associatedwith a cancer. “Cytotoxicity” refers to the ability of the cytotoxic Tlymphocyte to kill a target cell. Such antigen-specific cytotoxicity canbe at least about 3-fold, at least about 10-fold greater, at least about100-fold greater or more than cytotoxicity against a cell not presentingthe antigen not associated with the cancer. Antibody dependentcell-mediated cytotoxicity (ADCC) also includes activation of naturalkiller cells (“NK cells”) which mediate cell killing via antibodybinding. The IgG1 antibodies and antigen-binding fragments describedherein can mediate ADCC via NK cells through the binding of endoglin.

B. Methods of Making and Expressing Humanized Anti-Endoglin Antibodies

A chimeric monoclonal antibody has been developed that binds endoglin.This antibody is designated TRC105 (also known as c-SN6j).

In one aspect, the antibodies and antigen-binding fragments thereofdescribed herein were created by humanization of the V_(L) and V_(H)sequences of the chimeric monoclonal TRC105 antibody (SEQ ID NOS. 1 and39, respectively).

Humanized immunoglobulins, including humanized antibodies, have beenconstructed by means of genetic engineering. Most humanizedimmunoglobulins that have been previously described have comprised aframework that is identical to the framework of a particular humanimmunoglobulin chain (i.e., an acceptor or recipient), and three CDRsfrom a non-human (i.e., donor) immunoglobulin chain. As describedherein, humanization can also include criteria by which a limited numberof amino acids in the framework of a humanized immunoglobulin chain areidentified and chosen to be the same as the amino acids at thosepositions in the donor rather than in the acceptor, in order to increaseor maintain the affinity of an antibody comprising the humanizedimmunoglobulin chain.

The present invention is based in part on the model that twocontributing causes of the loss of affinity in prior means of producinghumanized antibodies (using as examples mouse antibodies as the sourceof CDRs) are: (1) when the mouse CDRs are combined with a humanframework, the amino acids in the frameworks close to the CDRs becomehuman instead of mouse. Without intending to be bound by theory, thesechanged amino acids may slightly distort the CDRs (e.g., they may createdifferent electrostatic or hydrophobic forces than in the donor mouseantibody, and the distorted-CDRs may not make as effective contacts withthe antigen as the CDRs did in the donor antibody); (2) also, aminoacids in the original mouse antibody that are close to, but not part of,the CDRs (i.e., still part of the framework), may make contacts with theantigen that contribute to affinity. These amino acids are lost when theantibody is humanized because, generally, all framework amino acids aremade human. To circumvent these issues, and to produce humanizedantibodies that have a very strong affinity for a desired antigen,humanized antibodies and antigen-binding fragments thereof can beconstructed using one or more of the following principles.

One non-limiting principle is that, for example, as acceptor, aframework is used from a particular human immunoglobulin that isunusually homologous to the donor immunoglobulin to be humanized, or usea consensus framework from many human antibodies is used as an acceptor.For example, comparison of the sequence of a mouse heavy (or light)chain variable region against human heavy (or light) variable regions ina data bank (for example, the National Biomedical Research FoundationProtein Identification Resource or the protein sequence database of theNational Center for Biotechnology Information—NCBI) shows that theextent of homology to different human regions can vary greatly, forexample from about 40% to about 60%, about 70%, about 80% or higher. Bychoosing as the acceptor immunoglobulin one of the human heavy chainvariable regions that is most homologous to the heavy chain variableregion of the donor immunoglobulin, fewer amino acids will be changed ingoing from the donor immunoglobulin to the humanized immunoglobulin. Bychoosing as the acceptor immunoglobulin one of the human light chainvariable regions that is most homologous to the light chain variableregion of the donor immunoglobulin, fewer amino acids will be changed ingoing from the donor immunoglobulin to the humanized immunoglobulin.Generally, using such techniques, there is a reduced chance of changingan amino acid near one or more of the CDRs that distorts theirconformation. Moreover, the precise overall shape of a humanizedantibody comprising the humanized immunoglobulin chain may more closelyresemble the shape of the donor antibody, thereby also reducing thechance of distorting the CDRs.

One can also use light and heavy chains from the same human antibody asacceptor sequences, to improve the likelihood that the humanized lightand heavy chains will make favorable contacts with each other.Alternatively, one can also use light and heavy chains from differenthuman antibody germline sequences as acceptor sequences; when suchcombinations are used, one can readily determine whether the V_(H) andV_(L) bind an epitope of interest using conventional assays (e.g., anELISA). In one example, the human antibody will be chosen in which thelight and heavy chain variable regions sequences, taken together, areoverall most homologous to the donor light and heavy chain variableregion sequences. Sometimes greater weight will be given to the heavychain sequence. Regardless of how the acceptor immunoglobulin is chosen,higher affinity can, in some cases, be achieved by selecting a smallnumber of amino acids in the framework of the humanized immunoglobulinchain to be the same as the amino acids at those positions in the donorrather than in the acceptor. Methods of affinity maturation are known inthe art.

Humanized antibodies generally have at least three potential advantagesover mouse or chimeric antibodies for use in human therapy. Because theeffector portion of an antibody is human, it is believed to interactbetter with the other parts of the human immune system (e.g., destroythe target cells more efficiently by complement-dependent cytotoxicity(CDC) or antibody-dependent cellular cytotoxicity (ADCC)). Additionally,the human immune system should not recognize the framework or constantregion of the humanized antibody as foreign, and therefore the antibodyresponse against such an injected antibody should be less than against atotally foreign mouse antibody or a partially foreign chimeric antibody.Finally, mouse antibodies are known to have a half-life in the humancirculation that is much shorter than the half-life of human antibodies.Humanized antibodies can, presumably, have a half-life more similar tonaturally-occurring human antibodies, allowing smaller and less frequentdoses to be given.

Humanization of antibodies and antigen-binding fragments thereof, can beaccomplished via a variety of methods known in the art and describedherein. Similarly, production of humanized antibodies can also beaccomplished via methods known in the art and described herein.

Methods for modifications of framework regions are known in the art andare contemplated herein. Selection of one or more relevant frameworkamino acid positions to be altered depends on a variety of criteria. Onecriterion for selecting relevant framework amino acids to change can bethe relative differences in amino acid framework residues between thedonor and acceptor molecules. Selection of relevant framework positionsto alter using this approach has the advantage of avoiding anysubjective bias in residue determination or any bias in CDR bindingaffinity contribution by the residue.

Another criterion that can be used for determining the relevant aminoacid positions to change can be, for example, selection of frameworkresidues that are known to be important or to contribute to CDRconformation. For example, canonical framework residues are importantfor CDR conformation and/or structure. Targeting of a canonicalframework residue as a relevant position to change can be used toidentify a more compatible amino acid residue in context with itsassociated donor CDR sequence.

The frequency of an amino acid residue at a particular frameworkposition is another criterion which can be used for selecting relevantframework amino acid positions to change. For example, comparison of theselected framework with other framework sequences within its subfamilycan reveal residues that occur at minor frequencies at a particularposition or positions. Positions harboring less abundant residues aresimilarly applicable for selection as a position to alter in theacceptor variable region framework.

The relevant amino acid positions to change also can be selected, forexample, based on proximity to a CDR. In certain contexts, FR residuescan participate in CDR conformation and/or antigen binding. Moreover,this criterion can similarly be used to prioritize relevant positionsselected by other criteria described herein. Therefore, differentiatingbetween residues proximal and distal to one or more CDRs represents oneway to reduce the number of relevant positions to change.

Other criteria for selecting relevant amino acid framework positions toalter include, for example, residues that are known or predicted toreside in a three dimensional space near the antigen-CDR interface orpredicted to modulate CDR activity. Similarly, framework residues thatare known to, or predicted to, form contacts between the heavy (V_(H))and light (V_(L)) chain variable region interface can be selected. Suchframework positions can affect the conformation and/or affinity of a CDRby modulating the CDR binding pocket, antigen (epitope) interaction orthe V_(H) and V_(L) interaction. Therefore, selection of these aminoacid positions for constructing a diverse population for screening ofbinding activity can be used to identify framework changes which replaceresidues having detrimental effects on CDR conformation or compensatefor detrimental effects of residues occurring elsewhere in theframework.

Other framework residues that can be selected for alteration includeamino acid positions that are inaccessible to solvent. Such residues aregenerally buried in the variable region and are, therefore, capable ofinfluencing the conformation of the CDR or V_(H) and V_(L) interactions.Solvent accessibility can be predicted, for example, from the relativehydrophobicity of the environment created by the amino acid side chainsof the polypeptide and/or by known three-dimensional structural data.

Following selection of relevant amino acid positions in the donor CDRs,as well as any relevant amino acid positions in the framework regionsdesired to be varied, amino acid changes at some or all of the selectedpositions can be incorporated into encoding nucleic acids for theacceptor variable region framework and donor CDRs. Altered framework orCDR sequences can be individually made and tested, or can besequentially or simultaneously combined and tested.

The variability at any or all of the altered positions can range from afew to a plurality of different amino acid residues, including alltwenty naturally occurring amino acids or functional equivalents andanalogues thereof. In some cases, non-naturally occurring amino acidsmay also be considered and are known in the art.

Selection of the number and location of the amino acid positions to varyis flexible and can depend on the intended use and desired efficiencyfor identification of the altered variable region having a desirableactivity such as substantially the same or greater binding affinitycompared to the donor variable region. In this regard, the greater thenumber of changes that are incorporated into an altered variable regionpopulation, the more efficient it is to identify at least one speciesthat exhibits a desirable activity, for example, substantially the sameor greater binding affinity as the donor. Alternatively, where the userhas empirical or actual data to the affect that certain amino acidresidues or positions contribute disproportionally to binding affinity,then it can be desirable to produce a limited population of alteredvariable regions which focuses on changes within or around thoseidentified residues or positions.

For example, if CDR grafted variable regions are desired, a large,diverse population of altered variable regions can include all thenon-identical framework region positions between the donor and acceptorframework and all single CDR amino acid position changes. Alternatively,a population of intermediate diversity can include subsets, for example,of only the proximal non-identical framework positions to beincorporated together with all single CDR amino acid position changesto, for example, increase affinity of the humanized antibodies orantigen binding fragments. The diversity of the above populations can befurther increased by, for example, additionally including all pair-wiseCDR amino acid position changes. In contrast, populations focusing onpredetermined residues or positions which incorporate variant residuesat as few as one framework and/or one CDR amino acid position cansimilarly be constructed for screening and identification of an alteredantibody variable region. As with the above populations, the diversityof such focused populations can be further increased by additionallyexpanding the positions selected for change to include other relevantpositions in either or both of the framework and CDR regions. There arenumerous other combinations ranging from few changes to many changes ineither or both of the framework regions and CDRs that can additionallybe employed, all of which will result in a population of alteredvariable regions that can be screened for the identification of at leastone CDR grafted altered variable region having desired activity, forexample, binding activity to endoglin. Those skilled in the art willknow, or can determine, which selected residue positions in theframework or donor CDRs, or subsets thereof, can be varied to produce apopulation for screening and identification of an altered antibody ofthe invention given the teachings and guidance provided herein. Codonsencoding amino acids are known in the art.

Another method of humanizing antibodies includes a method termed“superhumanization.” Superhumanization involves the steps of obtaining apeptide sequence for a subject variable region encoded by a non-humanmature antibody gene and identifying a first set of canonical CDRstructure types for at least two CDRs within the non-human antibodyvariable region. Canonical CDR structure types are the structure typesdesignated by Chothia (CITE). Chothia and coworkers found that criticalportions of the CDRs of many antibodies adopt nearly identical peptidebackbone conformations, despite great diversity at the level of aminoacid sequence. Accordingly, Chothia defined for each CDR in each chainone or a few “canonical structures.” Each canonical structure specifiesprimarily a set of peptide backbone torsion angles for a contiguoussegment of amino acid residues forming a loop.

After the identification of the canonical CDR structure type, a libraryof peptide sequences for human antibody variable regions for humanantibodies is also obtained. This library contains sequences for humangermline variable regions as encoded by germline nucleic acid segments,and may include mature human antibody sequences. In either case, themethod includes identifying canonical CDR structure types (i.e., asecond set of canonical CDR structure types) for at least two CDRs foreach sequence within the library of human variable region sequences.From this library there is selected a subset of candidate sequences bycomparing the first set of canonical CDR structure types to the secondset of canonical CDR structure types (i.e., comparing the mousecanonical CDR structure types to the human canonical CDR structure typesat corresponding locations within the variable region) and selectingthose human sequences where the second set of canonical CDR structure isthe same as the first set of canonical CDR structure types for the CDRsequences at corresponding locations within the non-human and humanvariable regions, respectively. The method uses these candidate humanvariable region sequences as a basis for constructing a chimericmolecule that includes at least two of the CDR sequences from thenon-human variable region (e.g., of the mouse CDRs) combined with theframework regions from candidate human variable region sequences. Theresult of the construction is that the chimeric antibody contains eachof the non-human CDR sequences substituted for each of the human CDRsequences at corresponding locations in the variable regions so that theframework sequences in the chimeric antibody differs from the candidatehuman framework sequences.

The similarity to the subject CDRs of candidate human antibody sequencesis assessed for each domain at two levels. Primarily, identicalthree-dimensional conformations of CDR peptide backbones are sought.Experimentally determined atomic coordinates of the subject CDRs areseldom available, hence three-dimensional similarity is approximated bydetermining Chothia canonical structure types of the subject CDRs andexcluding from further consideration candidates possessing differentcanonical structures. Secondarily, residue-to-residue homology betweensubject CDRs and the remaining human candidate CDRs is considered, andthe candidate with the highest homology is chosen.

Choosing highest homology is based on various criterion used to rankcandidate human variable regions having the same canonical structure asthe subject the non-human variable regions. The criterion for rankingmembers of the selected set may be by amino acid sequence identity oramino acid homology or both. Amino acid identity is simple a score ofposition by position matches of amino acid residues. Similarity by aminoacid homology is position by position similarity in residue structure ofcharacter. Homology may be scored, for example, according to the tablesand procedures described by Henikoff and Henikoff, (1992) Amino acidsubstitution matrices from protein blocks, Proc. Natl. Acad. Sci. USA89: 10915-10919, or by the BLOSUM series described by Henikoff andHenikoff, (1996). The steps are as follows:

a) Determine the peptide sequences of the heavy and light chain variabledomains of the subject antibody. These can be determined by any ofseveral methods, such as DNA sequencing of the respective genes afterconventional cDNA cloning; DNA sequencing of cloning products that havebeen amplified by the polymerase chain reaction from reverse transcriptsor DNA of the subject hybridoma line; or peptide sequencing of apurified antibody protein.

b) Apply the Kabat numbering system (Kabat et al, Id. 1991) to the heavyand light chain sequences of the subject non-human antibody. Determinecanonical structure types for each of the CDRs of the subject non-humanantibody. This determination is made from examination of the peptidesequence in light of the guidelines discussed in Chothia and Lesk(1987), Chothia et al. (1992), Tomlinson et al. (1995), Martin andThornton (1996), and Al-Lazikani et al. (1997).

The salient features of canonical structure determination for each ofthe CDRs are as follows. For heavy chain CDR1, three canonical structuretypes are currently known. Assignment of a new sequence isstraightforward because each canonical structure type has a differentnumber of residues. As described in Al-Lazikani et al. (1997), whenKabat numbering is assigned to the sequence, the numbering for residues31-35 will be as follows for the respective canonical structures:

Canonical structure type 1: 31, 32, 33, 34, 35;

Canonical structure type 2: 31, 32, 33, 34, 35, 35a; and

Canonical structure type 3: 31, 32, 33, 34, 35, 35a, 35b.

For heavy chain CDR2, four canonical structure types are currentlyknown. Several have unique numbers of residues, and are easilydistinguished from their unique Kabat numbering of positions 52-56,viz.:

Canonical structure type 1: 52, 53, 54, 55, 56; and

Canonical structure type 4: 52, 52a, 52b, 52c, 53, 54, 55, 56.

Canonical structure types 2 and 3 for heavy chain CDR2 have equalnumbers of residues, hence must be distinguished by clues within theirsequence, as discussed by Chothia et al. (1992). The Kabat numbering ofthe segment containing these clues is: 52, 52a, 53, 54, 55. Canonicalstructure type 2 has Pro or Ser at position 52a and Gly or Ser atposition 55, with no restriction at the other positions. Canonicalstructure type 3 has Gly, Ser, Asn, or Asp at position 54, with norestriction at the other positions. These criteria are sufficient toresolve the correct assignment in most cases. Additionally, frameworkresidue 71 is commonly Ala, Val, Leu, Ile, or Thr for canonicalstructure type 2 and commonly Arg for canonical structure type 3.

Heavy chain CDR3 is the most diverse of all the CDRs. It is generated bygenetic processes, some of a random nature, unique to lymphocytes.Consequently, canonical structures for CDR3 have been difficult topredict. In any case, human germline V gene segments do not encode anypart of CDR3; because the V gene segments end at Kabat position 94,whereas positions 95 to 102 encode CDR3. For these reasons, canonicalstructures of CDR3 are generally not considered for choosing candidatehuman sequences.

For light chain CDR1, six canonical structure types are currently knownfor CDR1 in kappa chains. Each canonical structure type has a differentnumber of residues, hence assignment of a canonical structure type to anew sequence is apparent from the Kabat numbering of residue positions27-31:

Canonical structure type 1: 27, 29, 30, 31;

Canonical structure type 2: 27, 28, 29, 30, 31;

Canonical structure type 3: 27, 27a, 27b, 27c, 27d, 27e, 27f, 28, 29,30, 31;

Canonical structure type 4: 27, 27a, 27b, 27c, 27d, 27e, 28, 29, 30, 31;

Canonical structure type 5: 27, 27a, 27b, 27c, 27d, 28, 29, 30, 31; and

Canonical structure type 6: 27, 27a, 28, 29, 30, 31.

For light chain CDR2, only a single canonical structure type is knownfor CDR2 in kappa chains, hence, barring exceptional subject antibodysequences, assignment is automatic. For light chain CDR3, up to sixcanonical structure types have been described for CDR3 in kappa chains,but three of these are rare. The three common ones can be distinguishedby their length, reflected in Kabat numbering of residue positions91-97:

Canonical structure type 1: 91, 92, 93, 94, 95, 96, 97 (also with anobligatory Pro at position 95 and Gln, Asn, or His at position 90):

Canonical structure type 3: 91, 92, 93, 94, 95, 97; and

Canonical structure type 5: 91, 92, 93, 94, 95, 96, 96a, 97.

After identifying the canonical CDR structure types of the subjectnon-human antibody, human genes of the same chain type (heavy or light)that have the same combination of canonical structure types as thesubject antibody are identified to form a candidate set of humansequences. Most of these gene fragments have been discovered and havealready been assigned to a canonical structure type (Chothia et al.,1992, Tomlinson et al., 1995).

For the heavy chain, conformity of CDR1 and CDR2 to the mouse canonicalstructure types is assessed, and genes that do not conform are excluded.For the light chain, conformity of CDR1 and CDR2 of each human sequenceto the canonical structure types of the subject antibody is firstassessed. The potential of residues 89-95 of a candidate Vk gene to forma CDR3 of the same canonical structure type as the subject antibody isassessed, by positing a fusion of the gene with a J region and applyingcriteria for CDR3 canonical CDR structure type determination to thefused sequence, and non-conforming sequences are excluded.

Alternatively, when a variable domain of the subject antibody is of acanonical structure type not available in the human genome, humangermline V genes that have three-dimensionally similar, but notidentical, canonical structure types are considered for comparison. Sucha circumstance often occurs with kappa chain CDR1 in murine antibodies,including two of the examples described below. All 6 possible canonicalstructure types have been observed at this CDR in murine antibodies,whereas the human genome encodes only canonical types 2, 3, 4 and 6. Inthese circumstances, a canonical CDR structure type having length ofamino acid residues within two of the length of the amino acid residuesof the subject non-human sequence may selected for the comparison. Forexample, where a type 1 canonical structure is found in the subjectantibody, human Vk sequences with canonical structure type 2 are usedfor comparison. Where a type 5 canonical structure is found in themurine antibody, human Vk sequences with either canonical structure type3 or 4 are be used for comparison.

Mature, rearranged human antibody sequences can be considered for thesequence comparison. Such consideration might be warranted under avariety of circumstances, including but not limited to instances wherethe mature human sequence (1) is very close to germline; (2) is knownnot to be immunogenic in humans; or (3) contains a canonical structuretype identical to that of the subject antibody, but not found in thehuman germline.

For each of the candidate V genes with matching canonical structuretypes, residue to residue sequence identity and/or homology with thesubject sequence is also evaluated to rank the candidate humansequences. For example, the residues evaluated are as follows: (1) Kappa(K) light chain CDR amino acid residue positions are CDR1 (26-32), CDR2(50-52), CDR3 (91-96); and (2) heavy chain CDR amino acid residuepositions are CDR1 (31-35) and CDR2 (50-60). Additionally, heavy chainCDR3 amino acid residue positions 95 to 102 can also be considered.

Residue-to-residue homology is first scored by the number of identicalamino acid residues between the subject and the candidate humansequences. The human sequence used for subsequent construction of aconverted antibody is chosen from among the 25 percent of candidateswith the highest score. When appropriate, such as when several candidatesequences have similar identity scores, similarity between non-identicalamino acid residues may be additionally considered as needed.Aliphatic-with-aliphatic, aromatic-with-aromatic, or polar-with-polarmatches between subject and object residues are added to the scores. Inanother example, quantitative evaluation of sequence homology may beperformed using amino acid substitution matrices such as the BLOSUM62matrix of Henikoff and Henikoff.

An object sequence for the framework region C-terminal to CDR3 sequencecan be selected from the set of known human germline J segments. A Jpeptide sequence is selected by evaluating residue to residue homologyfor each J segment for sequence positions for which CDR3 and J overlap,using the scoring criteria specified for the evaluation of candidate Vgenes as mentioned above. The J gene segment peptide sequence used forsubsequent construction of a converted antibody is chosen from among the25 percent of candidates with the highest score.

As an example, the chimeric variable chain contains at least two CDRsfrom a subject non-human sequence, and framework sequences from acandidate human sequence. In another example, chimeric light chaincontains three CDRs from a subject non-human sequence and frameworksequences from a candidate human sequence. In additional examples, achimeric heavy chain contains at least two CDRs of a subject heavychain, and framework sequence of a candidate human heavy chain, or achimeric heavy chin contains each of the CDRs from the subject heavychain and framework sequences of a candidate human heavy chain. In stillanother example, a chimeric antibody heavy chain contains CDRs 1 and 2from a subject non-human sequence and residues 50-60 for CDR3 andresidues 61-65 of a CDR from the candidate human heavy chain, along withthe framework sequences of the candidate human sequence. In anotherexample, a chimeric heavy chain sequence contains each CDR from thesubject non-human sequence; frameworks sequences 27-30 form the subjectsequence, and the framework sequences from the candidate sequences. Inall cases however, the chimeric antibody molecule contains no more than10 amino acid residues in the framework sequence that differ from thosein the framework sequence of the candidate human variable ration.

When increased affinity of a humanized antibody is desired, residueswithin the CDRs of a converted antibody may be additionally substitutedwith other amino acids. Typically, no more than four amino acid residuesin a CDR are changed, and most typically no more than two residues inthe CDR will be changed, except for heavy chain CDR2, where as many as10 residues may be changed. Changes in affinity can be measured byconventional methods such as those described herein (e.g., Biacore).

The methods of superhumanizing antibodies are described in more detailin U.S. Pat. No. 6,881,557 which is hereby incorporated by reference inits entirety.

Humanized antibodies and antigen-binding fragments can be constructedand produced using conventional techniques known in the art. Inaddition, recombinantly prepared antibodies can often be produced inlarge quantities, particularly when utilizing high level expressionvectors.

Antibodies can be sequenced using conventional techniques known in theart. In one aspect, the amino acid sequences of one or more of the CDRsis inserted into a synthetic sequence of, for example, a human antibody(or antigen-binding fragment thereof) framework to create a humanantibody that could limit adverse side reactions of treating a humanpatient with a non-human antibody. The amino acid sequences of one ormore of the CDRs can also be inserted into a synthetic sequence of, forexample, into a binding protein such as an AVIMER™ to create a constructfor administration to a human patient. Such techniques can be modifieddepending on the species of animal to be treated. For example, forveterinary uses, an antibody, antigen-binding fragment or bindingprotein can be synthesized for administration of a non-human (e.g., aprimate, a cow, a horse, etc.).

In another aspect, using art-recognized techniques such as thoseprovided and incorporated herein, nucleotides encoding amino acidsequences of one or more of the CDRs can inserted, for example, byrecombinant techniques in restriction endonuclease sites of an existingpolynucleotide that encodes an antibody, antigen-binding fragment orbinding protein.

For expression, an expression system is one which utilizes the GS system(Lonza) using a glutamine synthetase gene as the selectable marker.Briefly, a transfection is performed in CHO cells by electroporation(250V) using the GS system (Lonza) using the glutamine synthetase geneas the selectable marker. Wild type CHO cells are grown in DMEM (Sigma)containing 10% dialyzed Fetal Calf Serum (FCS) with 2 mM glutamine.6×10⁷ CHO cells are transfected with 300 μg of linearized DNA byelectroporation. After electroporation the cells are resuspended in DMEMwith glutamine and plated out into 36×96-well plates (50 μl/well), andincubated at 37° C. in 5% CO₂. The following day, 150 of selectivemedium (DMEM without glutamine) is added. After approximately 3 weeksthe colonies are screened by ELISA (see below) using an irrelevantantibody as a negative control. All colonies producing >20 μg/ml areexpanded into 24-well plates and then into duplicate T25 flasks.

For high level production, the most widely used mammalian expressionsystem is one which utilizes the gene amplification procedure offered bydihydrofolate reductase deficient (“dhfr-”) Chinese hamster ovary cells.The system is well known to the skilled artisan. The system is basedupon the dihydrofolate reductase “dhfr” gene, which encodes the DHFRenzyme, which catalyzes conversion of dihydrofolate to tetrahydrofolate.In order to achieve high production, dhfr-CHO cells are transfected withan expression vector containing a functional DHFR gene, together with agene that encodes a desired protein. In this case, the desired proteinis recombinant antibody heavy chain and/or light chain.

By increasing the amount of the competitive DHFR inhibitor methotrexate(MTX), the recombinant cells develop resistance by amplifying the dhfrgene. In standard cases, the amplification unit employed is much largerthan the size of the dhfr gene, and as a result the antibody heavy chainis co-amplified.

When large scale production of the protein, such as the antibody chain,is desired, both the expression level and the stability of the cellsbeing employed are taken into account. In long term culture, recombinantCHO cell populations lose homogeneity with respect to their specificantibody productivity during amplification, even though they derive froma single, parental clone.

The present application provides an isolated polynucleotide (nucleicacid) encoding an antibody or antigen-binding fragment as describedherein, vectors containing such polynucleotides, and host cells andexpression systems for transcribing and translating such polynucleotidesinto polypeptides.

The present application also provides constructs in the form ofplasmids, vectors, transcription or expression cassettes which compriseat least one polynucleotide as above.

The present application also provides a recombinant host cell whichcomprises one or more constructs as above. A nucleic acid encoding anyantibody or antigen-binding fragments thereof described herein asprovided itself forms an aspect of the present application, as does amethod of production of the antibody or antigen-binding fragmentsthereof described herein which method comprises expression from encodingnucleic acid therefrom. Expression can conveniently be achieved byculturing under appropriate conditions recombinant host cells containingthe nucleic acid. Following production by expression, an antibody orantigen-binding fragment can be isolated and/or purified using anysuitable technique, then used as appropriate. Antibodies describedherein have been engineered with particular features in mind to reducetheir immunogenicity to prevent human anti-mouse antibody (HAMA)reactions while retaining specificity and avidity.

Specific antibodies, antigen-binding fragments, and encoding nucleicacid molecules and vectors described herein can be provided isolatedand/or purified, e.g., from their natural environment, in substantiallypure or homogeneous form. In the case of nucleic acid, free orsubstantially free of nucleic acid or genes origin other than thesequence encoding a polypeptide with the required function. Nucleic acidcan comprise DNA or RNA and can be wholly or partially synthetic.Methods of purification are well known in the art.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known. Suitable host cells includebacterial cells, mammalian cells, yeast cells and baculovirus systems.Mammalian cell lines available in the art for expression of aheterologous polypeptide include Chinese hamster ovary cells, HeLacells, baby hamster kidney cells, NS0 mouse myeloma cells and manyothers. A common bacterial host is E. coli.

The expression of antibodies and antibody fragments in prokaryotic cellssuch as E. coli is well established in the art. For a review, see, forexample Plückthun, A., Bio/Technology 9: 545-551 (1991). Expression ineukaryotic cells in culture is also available to those skilled in theart as an option for production of the antibodies and antigen-bindingfragments described herein, see for recent reviews, for example Raff, M.E., (1993) Curr. Opinion Biotech. 4: 573-576; Trill J. J., et al. (1995)Curr. Opinion Biotech 6: 553-560, each of which is which is incorporatedherein by reference in its entirety.

Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorsequences, polyadenylation sequences, enhancer sequences, marker genesand other sequences as appropriate. Vectors can be plasmids, viral e.g.,‘phage, or phagemid, as appropriate. For further details see, forexample, Molecular Cloning: a Laboratory Manual: 2nd Ed., Sambrook etal., 1989, Cold Spring Harbor Laboratory Press. Many known techniquesand protocols for manipulation of nucleic acid, for example inpreparation of nucleic acid constructs, mutagenesis, sequencing,introduction of DNA into cells and gene expression, and analysis ofproteins, are described in detail in Short Protocols in MolecularBiology, Second Edition, Ausubel et al. Eds., John Wiley & Sons, 1992.The methods disclosures of Sambrook et al. and Ausubel et al. areincorporated herein by reference in their entirety and are well known inthe art.

Thus, a further aspect provides a host cell containing nucleic acid asdisclosed herein. A still further aspect provides a method comprisingintroducing such nucleic acid into a host cell. The introduction canemploy any available technique. For eukaryotic cells, suitabletechniques can include, for example, calcium phosphate transfection,DEAE Dextran, electroporation, liposome-mediated transfection andtransduction using retrovirus or other virus, e.g., vaccinia or, forinsect cells, baculovirus. For bacterial cells, suitable techniques caninclude, for example, calcium chloride transformation, electroporationand transfection using bacteriophage.

The introduction can be followed by causing or allowing expression fromthe nucleic acid, e.g., by culturing host cells under conditions forexpression of the gene.

In one embodiment, the nucleic acid is integrated into the genome (e.g.,chromosome) of the host cell. Integration can be promoted by inclusionof sequences which promote recombination with the genome, in accordancewith standard techniques. Ig enhances can be initialized as needed tomaximize expression.

The present application also provides a method which comprises using aconstruct as stated above in an expression system in order to expressthe antibodies or antigen-binding fragments thereof as above.

The present application also relates to isolated nucleic acids, such asrecombinant DNA molecules or cloned genes, or degenerate variantsthereof, mutants, analogs, or fragments thereof, which encode anantibody or antigen-binding sequence described herein that bindsendoglin.

In one aspect, the present application provides a nucleic acid whichcodes for an antibody or antigen-binding fragment thereof as describedherein which binds endoglin.

In a further embodiment, the full DNA sequence of the recombinant DNAmolecule or cloned gene of an antibody or antigen-binding fragmentdescribed herein can be operatively linked to an expression controlsequence which can be introduced into an appropriate host. Theapplication accordingly extends to unicellular hosts transformed withthe cloned gene or recombinant DNA molecule comprising a DNA sequenceencoding the V_(H) and/or V_(L), or portions thereof, of the antibody.

Another feature is the expression of the DNA sequences disclosed herein.As is well known in the art, DNA sequences can be expressed byoperatively linking them to an expression control sequence in anappropriate expression vector and employing that expression vector totransform an appropriate unicellular host.

Such operative linking of a DNA sequence to an expression controlsequence, of course, includes, if not already part of the DNA sequence,the provision of an initiation codon, ATG, in the correct reading frameupstream of the DNA sequence.

Polynucleotides and vectors can be provided in an isolated and/or apurified form (e.g., free or substantially free of polynucleotides oforigin other than the polynucleotide encoding a polypeptide with therequired function). As used herein, “substantially pure,” and“substantially free” refer to a solution or suspension containing lessthan, for example, about 20% or less extraneous material, about 10% orless extraneous material, about 5% or less extraneous material, about 4%or less extraneous material, about 3% or less extraneous material, about2% or less extraneous material, or about 1% or less extraneous material.

A wide variety of host/expression vector combinations can be employed inexpressing the DNA sequences of this invention. Useful expressionvectors, for example, can consist of segments of chromosomal,non-chromosomal and synthetic DNA sequences. Suitable vectors include,but are not limited to, derivatives of SV40 and known bacterialplasmids, e.g., E. coli plasmids col E1, Pcr1, Pbr322, Pmb9 and theirderivatives, plasmids such as RP4; phage DNAs, e.g., the numerousderivatives of phage k, e.g., NM989, and other phage DNA, e.g., M13 andfilamentous single stranded phage DNA; yeast plasmids such as the 2uplasmid or derivatives thereof; vectors useful in eukaryotic cells, suchas vectors useful in insect or mammalian cells; vectors derived fromcombinations of plasmids and phage DNAs, such as plasmids that have beenmodified to employ phage DNA or other expression control sequences; andthe like.

Also provided herein is a recombinant host cell which comprises one ormore polynucleotide constructs. A polynucleotide encoding an antibody orantigen-binding fragment as provided herein forms an aspect of thepresent application, as does a method of production of the antibody orantigen-binding fragment which method comprises expression from thepolynucleotide. Expression can be achieved, for example, by culturingunder appropriate conditions recombinant host cells containing thepolynucleotide. An antibody or antigen-binding fragment can then beisolated and/or purified using any suitable technique, and used asappropriate.

Any of a wide variety of expression control sequences—sequences thatcontrol the expression of a DNA sequence operatively linked to it—can beused in these vectors to express the DNA sequences. Such usefulexpression control sequences include, for example, the early or latepromoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lac system,the trp system, the TAC system, the TRC system, the LTR system, themajor operator and promoter regions of phage λ, the control regions offd coat protein, the promoter for 3-phosphoglycerate kinase or otherglycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5), thepromoters of the yeast alpha-mating factors, and other sequences knownto control the expression of genes of prokaryotic or eukaryotic cells ortheir viruses, and various combinations thereof.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known. Suitable host cells includebacteria, mammalian cells, yeast and baculovirus systems. Mammalian celllines available in the art for expression of a heterologous polypeptideinclude Chinese hamster ovary (CHO) cells, HeLa cells, baby hamsterkidney cells, NS0 mouse myeloma cells and many others. A common,bacterial host can be, for example, E. coli.

The expression of antibodies or antigen-binding fragments in prokaryoticcells, such as E. coli, is well established in the art. For a review,see for example Plückthun, A., Bio/Technology 9: 545-551 (1991).Expression in eukaryotic cells in culture is also available to thoseskilled in the art (Raff, M. E., (1993) Curr. Opinion Biotech. 4:573-576; Trill J. J. et al., (1995) Curr. Opinion Biotech 6: 553-560).

A wide variety of unicellular host cells are also useful in expressingthe DNA sequences. These hosts include well-known eukaryotic andprokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus,Streptomyces, fungi such as yeasts, and animal cells, such as CHO,YB/20, NS0, SP2/0, R1.1, B-W and L-M cells, African Green Monkey kidneycells (e.g., COS 1, COS 7, BSC1, BSC40, and BMT10), insect cells (e.g.,Sf9), and human cells and plant cells in tissue culture.

It will be understood that not all vectors, expression control sequencesand hosts will function equally well to express the DNA sequences.Neither will all hosts function equally well with the same expressionsystem. However, one skilled in the art will be able to select theproper vectors, expression control sequences, and hosts without undueexperimentation to accomplish the desired expression without departingfrom the scope of this application. For example, in selecting a vector,the host must be considered because the vector must function in it. Thevector's copy number, the ability to control that copy number, and theexpression of any other proteins encoded by the vector, such asantibiotic markers, will also be considered. One of ordinary skill inthe art can select the proper vectors, expression control sequences, andhosts to accomplish the desired expression without departing from thescope of this application. For example, in selecting a vector, the hostis considered because the vector functions in it. The vector's copynumber, the ability to control that copy number, and the expression ofany other proteins encoded by the vector, such as antibiotic markers,can also be considered.

The present application also provides constructs in the form ofplasmids, vectors, transcription or expression cassettes as describedelsewhere herein which comprise at least one polynucleotide as above.Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorsequences, polyadenylation sequences, enhancer sequences, selectablemarkers and other sequences as appropriate. Vectors can be plasmids,viral e.g., phage, phagemid, etc., as appropriate. For further detailssee, for example, Molecular Cloning: a Laboratory Manual: 2nd Ed.,Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. Many knowntechniques and protocols for manipulation of nucleic acid, for examplein preparation of nucleic acid constructs, mutagenesis, sequencing,introduction of DNA into cells and gene expression, and analysis ofproteins, are described in detail in Short Protocols in MolecularBiology, Second Edition, Ausubel et al. Eds., John Wiley & Sons, 1992.The methods and disclosures of Sambrook et al. and Ausubel et al. areincorporated herein by reference.

In selecting an expression control sequence, a variety of factors willnormally be considered. These include, for example, the relativestrength of the system, its controllability, and its compatibility withthe particular DNA sequence or gene to be expressed, particularly asregards potential secondary structures. Suitable unicellular hosts willbe selected by consideration of, e.g., their compatibility with thechosen vector, their secretion characteristics, their ability to foldproteins correctly, and their fermentation requirements, as well as thetoxicity to the host of the product encoded by the DNA sequences to beexpressed, and the ease of purification of the expression products.

A further aspect provides a host cell containing one or morepolynucleotides as disclosed herein. Yet a further aspect provides amethod of introducing such one or more polynucleotides into a host cell,any available technique. For eukaryotic cells, suitable techniques caninclude, for example, calcium phosphate transfection, DEAE Dextran,electroporation, liposome-mediated transfection and transduction usingretrovirus or other virus (e.g., vaccinia) or, for insect cells,baculovirus. For bacterial cells, suitable techniques can include, forexample calcium chloride transformation, electroporation andtransfection using bacteriophages.

The introduction can be followed by causing or allowing expression fromthe one or more polynucleotides, e.g., by culturing host cells underconditions for expression of one or more polypeptides from one or morepolynucleotides. Inducible systems can be used and expression induced byaddition of an activator.

In one embodiment, the polynucleotides can be integrated into the genome(e.g., chromosome) of the host cell. Integration can be promoted byinclusion of sequences which promote recombination with the genome, inaccordance with standard techniques. In another embodiment, the nucleicacid is maintained on an episomal vector in the host cell.

Methods are provided herein which include using a construct as statedabove in an expression system in order to express a specificpolypeptide.

Considering these and other factors, a person skilled in the art will beable to construct a variety of vector/expression control sequence/hostcombinations that will express the DNA sequences on fermentation or inlarge scale animal culture.

A polynucleotide encoding an antibody, antigen-binding fragment, or abinding protein can be prepared recombinantly/synthetically in additionto, or rather than, cloned. The polynucleotide can be designed with theappropriate codons for the antibody, antigen-binding fragment, or abinding protein. In general, one will select preferred codons for anintended host if the sequence will be used for expression. The completepolynucleotide can be assembled from overlapping oligonucleotidesprepared by standard methods and assembled into a complete codingsequence. See, e.g., Edge, Nature, 292:756 (1981); Nambair et al.,Science, 223:1299 (1984); Jay et al., J. Biol. Chem., 259:6311 (1984).

A general method for site-specific incorporation of unnatural aminoacids into proteins is described in Christopher J. Noren, Spencer J.Anthony-Cahill, Michael C. Griffith, Peter G. Schultz, Science,244:182-188 (April 1989). This method can be used to create analogs withunnatural amino acids.

As mentioned above, a DNA sequence encoding an antibody orantigen-binding fragment thereof can be prepared synthetically ratherthan cloned. The DNA sequence can be designed with the appropriatecodons for the antibody or antigen-binding fragment amino acid sequence.In general, one will select preferred codons for the intended host ifthe sequence will be used for expression. The complete sequence isassembled from overlapping oligonucleotides prepared by standard methodsand assembled into a complete coding sequence. See, e.g., Edge, Nature,292:756 (1981); Nambair et al., Science, 223:1299 (1984); Jay et al., J.Biol. Chem., 259:6311 (1984), each of which is which is incorporatedherein by reference in its entirety.

C. In Silico Analysis of Immunogenicity

If needed, an antibody or an antigen binding fragment thereof describedherein can be assessed for immunogenicity and, as needed, be deimmunized(i.e., the antibody is made less immuno reactive by altering one or moreT cell epitopes). Analysis of immunogenicity and T-cell epitopes presentin the humanized anti-endoglin antibodies and antigen-binding fragmentsdescribed herein can be carried out via the use of software and specificdatabases. Exemplary software and databases include iTope™ developed byAntitope of Cambridge, England. iTope™ is an in silico technology foranalysis of peptide binding to human MHC class II alleles.

The iTope™ software predicts peptide binding to human MHC class IIalleles and thereby provides an initial screen for the location of such“potential T cell epitopes.” iTope™ software predicts favorableinteractions between amino acid side chains of a peptide and specificbinding pockets within the binding grooves of 34 human MHC class IIallelles. The location of key binding residues is achieved by the insilico generation of 9 mer peptides that overlap by one amino acidspanning the test antibody variable region sequence. Each 9 mer peptidecan be tested against each of the 34 MHC class II allotypes and scoredbased on their potential “fit” and interactions with the MHC class IIbinding groove. Peptides that produce a high mean binding score (>0.55in the iTope™ scoring function) against >50% of the MHC class II allelesare considered as potential T cell epitopes. In such regions, the core 9amino acid sequence for peptide binding within the MHC class II grooveis analyzed to determine the MHC class II pocket residues (P1, P4, P6,P7 and P9) and the possible T cell receptor (TCR) contact residues (P-1,P2, P3, P5, P8).

After identification of any T-cell epitopes, amino acid residue changes,substitutions, additions, and/or deletions can be introduced to removethe identified T-cell epitope. Such changes can be made so as topreserve antibody structure and function while still removing theidentified epitope. Exemplary changes can include, but are not limitedto, conservative amino acid changes.

Techniques exploiting soluble complexes of recombinant MHC molecules incombination with synthetic peptides have come into use. These reagentsand procedures may be used to identify the presence of T-cell clonesfrom peripheral blood samples from human or experimental animal subjectsthat are able to bind particular MHC-peptide complexes and are notadapted for the screening multiple potential epitopes to a widediversity of MHC allotypes.

Biological assays of T-cell activation remain the best practical optionto providing a reading of the ability of a test peptide/protein sequenceto evoke an immune response. Examples of this kind of approach includethe use of T-cell proliferation assays to the bacterial proteinstaphylokinase, followed by epitope mapping using synthetic peptides tostimulate T-cell lines. Similarly, T-cell proliferation assays usingsynthetic peptides of the tetanus toxin protein have resulted indefinition of immunodominant epitope regions of the toxin. In oneembodiment, T-cell epitopes in a test protein may be determined usingisolated sub-sets of human immune cells, promoting their differentiationin vitro and culture of the cells in the presence of synthetic peptidesof interest and measurement of any induced proliferation in the culturedT-cells. Other techniques may also be used. Such a technique involvescareful application of cell isolation techniques and cell culture withmultiple cytokine supplements to obtain the desired immune cell sub-sets(dendritic cells, CD4+ and or CD8+ T-cells). In another embodiment, thepresence of T cell epitopes in an antibody may be determined by addingthe antibody to isolated sub-sets of human immune cells, and assessingtheir differentiation in vitro and measuring any induced proliferationin the cultured T cells.

In silico techniques to define MHC class II ligands for multipleproteins of therapeutic interest may also be utilized. However, forreasons such as the requirement for proteolytic processing and otherphysiologic steps leading to the presentation of immunogenic peptides invivo, a sub-set of the entire repertoire of peptides definable bycomputer-based schemes may have ultimate biological relevance. Thus, exvivo human T-cell activation assays may be used to identify the regionswithin the protein sequence of a polypeptide that are able to supportT-cell activation and are thereby most biologically relevant to theproblem of immunogenicity in this protein. As used herein, “T-cellepitope” refers to an amino acid sequence which is able to bind MHCclass II, able to stimulate T-cells and/or also to bind (withoutnecessarily measurably activating) T-cells in complex with MHC class II.

According to a method disclosed herein, synthetic peptides or wholeantibodies are tested for their ability to evoke a proliferativeresponse in human T-cells cultured in vitro. The T-cells are presentwithin a peripheral blood mononuclear cell (PBMC) layer readilyobtainable by well known means from whole blood samples. Moreover, thePBMC preparation contains physiological ratios of T-cells and antigenpresenting cells and is, therefore, a good source of materials withwhich to conduct a surrogate immune reaction in vitro. In the operationof such an assay, a stimulation index approaching or exceeding 2.0 is auseful measure of induced proliferation. However, the stimulation indexmay be different depending upon the antibody, or antigen-bindingfragment thereof, and may be established with reference to a baselinefor each antibody, or antigen-binding fragment thereof, andcorresponding peptide library. In one example of such testing, thestimulation index (SI) may be conventionally derived by division of theproliferation score (e.g., counts per minute of radioactivity if usingfor example ³H-thymidine incorporation) measured to the test peptide bythe score measured in cells not contacted with a test peptide. Peptideswhich evoke no response may give a SI=1.0 although SI values in therange 0.8-1.2 may also be unremarkable. A number of technical procedurescan be built into the operation of such assays in order to ensureconfidence in the recorded scores. Typically all determinations are madeat least in triplicate and the mean score may be computed. Where acomputed SI=>2.0, individual scores of the triplicate can be examinedfor evidence of outlying data. Test peptides are contacted with cells inat least two different concentrations and the concentrations wouldtypically span a minimum two-fold concentration difference. Such aconcentration range provides an off-set to the kinetic dimension to theassay and may be useful where a single time point determination, forexample at day plus 7, is being conducted. In some assays, multiple timecourse determinations may be conducted and these too may be made usingpeptide immunogen provided at a minimum of two different concentrations.Similarly the inclusion of control peptides for which there isexpectation that the majority of PBMC donor samples will be responsivemay be included in each assay plate. The influenza haemagglutininpeptide 307-309, sequence PKYVKQNTLKLA (SEQ ID NO: 104); and theChlamydia HSP 60 peptide sequence KVVDQIKKISKPVQH (SEQ ID NO: 105) areexamples of control peptides to be used in such an assay. Alternatively,or in addition, assays could also use a potent whole protein antigen,such as hemocyanin from Keyhole Limpet, to which all PBMC samples wouldbe expected to exhibit an SI significantly greater than 2.0. Othercontrol antigens for such use will be well-known in the art.

The methods disclosed herein can provide an epitope map of antibodies,or antigen-binding fragments thereof, where the map has relevance to awide spectrum of possible MHC allotypes. The map may be sufficientlyrepresentative to allow the design or selection of a modified proteinfor which the ability of the protein to evoke a T-cell driven immuneresponse may be eliminated or at least ameliorated for the majority ofpatients to whom the protein is likely to be administered. Ameliorationcan refer to a reduction in an immune response (i.e., reducedimmunogenicity) compared to an unmodified protein (e.g., about 1.5 foldless, about 2 fold less, about 5 fold less, about 10 fold less, about 20fold less, about 50 fold less, about 100 fold less, about 200 fold less,about 500 fold less or more, or any range therein). Alternatively,antibodies, or antigen-binding fragments thereof, with reducedimmunogenicity can refer to a percent reduction in its ability to elicitan immune response compared to an unmodified protein (e.g., about 1%less, about 2% less, about 3% less, about 4% less, about 5% less, about10% less, about 20% less, about 50% less, about 100% less, and any rangetherein). Accordingly in the practice of the screening process, PBMCderived T-cells from naive donors are collected from a pool of donors ofsufficient immunological diversity to provide a sample of at leastgreater than 90% of the MHC class II repertoire (HLA-DR) extant in thehuman population. Where a naive T-cell response is to be detected to agiven synthetic peptide (or antibody), the peptide (or antibody) inpractice is contacted with PBMC preparations derived from multipledonors in isolation; the numbers of donors (or “donor pool” size), isfor practical purposes not likely to be less than 20 unrelatedindividuals and all samples in the donor pool may be pre-selectedaccording to their MHC class II haplotype.

As used herein, the term “naive donor” refers to a subject that has notbeen previously exposed to antibodies, or antigen-binding fragmentsthereof, described herein either environmentally, by vaccination, or byother means such as, for example, blood transfusions.

When screening for T-cell epitopes, T-cells can be provided from aperipheral blood sample from a multiplicity of different healthy donorsbut who have not been in receipt of the protein therapeutically. Ifneeded, patient blood samples can be tested for the presence of aparticular polypeptide using conventional assays such as an ELISA whichuses antibodies to identify the presence or absence of one or morepolypeptides. The assay is conducted using PBMC cultured in vitro usingconventional procedures known in the art and involves contacting thePBMC with synthetic peptide species representative of the protein ofinterest (i.e., a library), or a whole protein, such as an antibody andfollowing a suitable period of incubation, measurement of induced T cellactivation such as cellular proliferation. Measurement can be by anysuitable means and may, for example, be conducted using H³-thymidineincorporation whereby the accumulation of H³ into cellular material isreadily measured using laboratory instruments. The degree of cellularproliferation for each combination of PBMC sample and synthetic peptideor whole protein can be examined relative to that seen in an untreatedPBMC sample. Reference may also be made to the proliferative responseseen following treatment with a peptide or peptides or whole proteinsfor which there is an expected proliferative effect. In this regard, itis advantageous to use a peptide or whole protein with known broad MHCrestriction and especially peptide epitopes with MHC restriction to theDP or DQ isotypes, although the invention is not limited to the use ofsuch restricted peptides or proteins. Such peptides have been describedabove, for example, with respect to influenza haemagglutinin andchlamydia HSP60.

In one non-limiting example, T-cell epitopes are mapped and subsequentlymodified using the methods described herein. To facilitate assembly ofan epitope map, a library of synthetic peptides is produced. Each of thepeptides is 15 amino acid residues in length and each overlapped thenext peptide in the series by 12 amino acid residues; i.e., eachsuccessive peptide in the series incrementally added a further 3 aminoacids to the analysis. In this way, any given adjacent pair of peptidesmapped 18 amino acids of contiguous sequence. One method for defining aT-cell map using naive T-cell assays is illustrated in the Examplesbelow. Each of the peptides identified via the method to define a T-cellmap are suggested to be able to bind MHC class II and engage at leastone cognate TCR with sufficient affinity to evoke a proliferative burstdetectable in the assay system.

In another non-limiting example, the potential of an antibody to beprocessed to generate T-cell epitopes that bind MHC class II and engageat least one cognate TCR with sufficient affinity to evoke aproliferative burst detectable in the assay system is assessed.

The molecules described herein can be prepared in any of several waysincluding the use of recombinant methods. The protein sequences andinformation provided herein can be used to deduce a polynucleotide (DNA)encoding an amino acid sequence. This can be achieved for example usingcomputer software tools such as the DNASTAR® software suite [DNASTAR®Inc, Madison, Wis., USA] or similar. Any such polynucleotide encodingthe polypeptides or significant homologues, variants, truncations,elongations, or further modifications thereof, are contemplated herein.

Provided herein are methods of mapping (identifying) T-cell epitopes andmodifying the epitopes such that the modified sequence reduces(partially or completely) induction of a T-helper response. Modificationincludes amino acid substitutions, deletions, or insertion made incodons of a polynucleotide encoding modified polypeptides to affectsimilar changes. Codons encoding amino acid residues are well known inthe art. It is possible to use recombinant DNA methods to achievedirected mutagenesis of the target sequences and many such techniquesare available, described herein, and known in the art such as describedabove. In general, the technique of site-specific mutagenesis is wellknown. Briefly, a bacteriophage vector that produces a single strandedtemplate for oligonucleotide directed PCR mutagenesis is employed. Phagevectors (e.g., M13) are commercially available and their use isgenerally well known in the art. Similarly, double stranded plasmids arealso routinely employed in site directed mutagenesis, which eliminatesthe step of transferring the polynucleotide of interest from a phage toa plasmid. Synthetic oligonucleotide primers bearing the desired mutatedsequence can be used to direct the in vitro synthesis of modified(desired mutant) DNA from this template and the heteroduplex DNA is usedto transform competent E. coli for the growth selection andidentification of desired clones. Alternatively, a pair of primers canbe annealed to two separate strands of a double stranded vector tosimultaneously synthesize both corresponding complementary strands withthe desired mutation(s) in a PCR reaction.

In one embodiment, the Quick Change site-directed mutagenesis methodusing plasmid DNA templates may be employed. PCR amplification of theplasmid template containing the insert target gene of insert is achievedusing two synthetic oligonucleotide primers containing the desiredmutation. The oligonucleotide primers, each complementary to oppositestrands of the vector, are extended during temperature cycling bymutagenesis-grade Pfu Turbo DNA polymerase. On incorporation of theoligonucleotide primers, a mutated plasmid containing staggered nicks isgenerated. Amplified un-methylated products are treated with Dpn I todigest methylated parental DNA template and select for the newlysynthesized DNA containing mutations. Since DNA isolated from most E.coli strains is dam methylated, it is susceptible to Dpn I digestion,which is specific for methylated and hemi-methylated DNA. The reactionproducts are transformed into high efficiency strains of E. coli toobtain plasmids containing the desired modifications. Additional methodsfor introducing amino acid modifications into a polypeptide are wellknown in the art and can also be used herein.

Suitable modifications to a protein may include amino acid substitutionof particular residues or combinations of residues. For the eliminationof T-cell epitopes, amino acid substitutions are made at appropriatepoints or amino acid residues within an amino acid sequence predicted toachieve reduction or elimination of the activity of the T-cell epitope.In practice, an appropriate point or amino acid residue will preferablyequate to an amino acid residue binding within one of the pocketsprovided within the MHC class II binding groove. Such modifications mayalter binding within the first pocket of the cleft at the so-called “P1”or “P1 anchor” position of the peptide. The quality of bindinginteraction between the P1 anchor residue of the peptide and the firstpocket of the MHC class II binding groove is recognized as being a majordeterminant of overall binding affinity for the whole peptide. Anappropriate substitution at this position of the amino acid sequencewill generally incorporate an amino acid residue less readilyaccommodated within the pocket (e.g., substitution to a more hydrophilicresidue). Amino acid residues in the peptide at positions equating tobinding within other pocket regions within the MHC binding cleft arealso considered and fall under the scope of the present.

It is understood that single amino acid modifications within a givenpotential T-cell epitope represent one route by which one or more T-cellepitopes may be eliminated. Combinations of modifications within asingle epitope may be contemplated and can be appropriate whereindividually defined epitopes are in overlap with each other. Moreover,amino acid modifications (either singly within a given epitope or incombination within a single epitope) may be made at positions notequating to the “pocket residues” with respect to the MHC class IIbinding groove, but at any point within the amino acid sequence.Modifications may be made with reference to a homologous structure orstructural method produced using in silico techniques known in the artand described herein may be based on known structural features of thepolypeptide. A change (modification) may be contemplated to restorestructure or biological activity of the variant molecule. Suchcompensatory changes and changes may also include deletion or addition(insertion) of particular amino acid residues from a polypeptide.Additionally, modifications can be made that alter the structure and/orreduce the biological activity of the molecule and also eliminate aT-cell epitope, thus reducing the immunogenicity of the molecule. Alltypes of modifications are contemplated herein.

An additional means of removing epitopes from protein molecules is theconcerted use of a naive T-cell activation assay scheme as outlinedherein together with an in silico tool developed according to the schemedescribed in WO 02/069232 which is also incorporated fully herein byreference. The software simulates the process of antigen presentation atthe level of the polypeptide-MHC class II binding interaction to providea binding score for any given polypeptide sequence. Such a score isdetermined for many of the predominant MHC class II allotypes extant inthe population. As this scheme is able to test any polypeptide sequence,the consequences of amino acid substitutions additions or deletions withrespect to the ability of a polypeptide to interact with a MHC class IIbinding groove can be predicted. Consequently new sequence compositionscan be designed which contain reduced numbers of amino acids able tointeract with a MHC class II and thereby function as immunogenic T-cellepitopes. Where the biological assay using any one given donor samplecan assess binding to a maximum of four DR allotypes, the in silicoprocess can test a same polypeptide sequence using >40 allotypessimultaneously. In practice this approach is able to direct the designof new sequence variants which are altered in their ability to interactwith multiple MHC allotypes. As will be clear to one in the art,multiple alternative sets of substitutions could be arrived at whichachieve the objective of removing undesired epitopes. The resultingsequences would however be recognized to be closely homologous with thespecific compositions disclosed herein and therefore fall within thescope of the present application.

A combined approach of using an in silico tool for the identification ofMHC class II ligands and design of sequence analogues lacking MHC classII ligands, in concert with epitope mapping and re-testing optionallyusing biologically based assays of T-cell activation is an additionalmethod and embodiment of the present application. The general methodaccording to this embodiment comprises the following steps:

i) use of naive T-cell activation assays and synthetic peptidescollectively encompassing the protein sequence of interest to identifyepitope regions capable of activating T-cells;

ii) use of a computational scheme simulating the binding of the peptideligand with one or more MHC allotypes to analyze the epitope regionsidentified in step (i) and thereby identify MHC class II ligands withinthe epitope region;

iii) use of a computational scheme simulating the binding of the peptideligand with one or more MHC allotypes to identify sequence analogues ofthe MHC ligands encompassed within the epitope region(s) which no longerbind MHC class II or bind with lowered affinity to a lesser number ofMHC allotypes and, optionally,

iv) use of naive T-cell activation assays and synthetic peptidesencompassing entirely or in collection encompassing the epitope regionsidentified within the protein of interest and testing the sequenceanalogues in naive T-cell activation assay in parallel with thewild-type (parental) sequences.

In one embodiment, a method of making a modified antibody, orantigen-binding fragment thereof, exhibiting reduced immunogenicitycompared to an unmodified antibody, or antigen-binding fragment thereof,comprises identifying at least one T-cell epitope within the amino acidsequence of an antibody, or antigen-binding fragment thereof, andmodifying at least one amino acid residue within at least one identifiedT-cell epitope.

In another embodiment, a modified antibody, or antigen-binding fragmentthereof, exhibiting reduced immunogenicity compared to an unmodifiedantibody, or antigen-binding fragment thereof, is produced by a processof identifying at least one T-cell epitope within the amino acidsequence of an antibody, or antigen-binding fragment thereof, andmodifying at least epitope one amino acid residue within at least oneidentified T-cell epitope.

In yet another embodiment, a method of selecting a modified antibody, orantigen-binding fragment thereof, that exhibits reduced immunogenicitycompared to an unmodified antibody, or antigen-binding fragment thereof,comprises identifying at least one T-cell epitope within the amino acidsequence of a antibody, or antigen-binding fragment thereof, modifyingat least one amino acid residue within at least one identified T-cellepitope, and selecting a modified antibody, or antigen-binding fragmentthereof, that exhibits reduced immunogenicity compared to an unmodifiedantibody, or antigen-binding fragment thereof.

T-cell epitopes described herein can be further characterized by theregions of the. Such regions include the epitope core, the N-terminusand the C-terminus. As used herein “epitope core” refers to the core9-mer amino acid sequences of the T-cell epitopes. The epitope core canfurther include 0, 1, 2, or 3 amino acid residues adjacent to the core9-mer amino acid sequence on the N-terminus and/or the C-terminus. Thusthe epitope core, in certain embodiments, can range in length from about9 amino acids up to about 15 amino acids.

As used herein, “N-terminus” refers to the amino acids adjacent to theN-terminus of the epitope core and includes at least 1, 2, 3, 4, 5, 6,7, 8 or 9 amino acids adjacent to and upstream of the N-terminus of theepitope core.

As used herein, “C-terminus” refers to the amino acids adjacent to theC-terminus of the epitope core and includes at least 1, 2, 3, 4, 5, 6,7, 8, or 9 amino acids adjacent to and downstream of the C-terminus ofthe epitope core.

In one embodiment, a modified antibody, or antigen-binding fragmentthereof, contains one or more modifications. In one embodiment, amodified antibody, or antigen-binding fragment thereof, contains twomodifications. In one embodiment, a modified antibody, orantigen-binding fragment thereof, contains three modifications. In oneembodiment, a modified antibody, or antigen-binding fragment thereof,contains four modifications. In one embodiment, a modified antibody, orantigen-binding fragment thereof, contains five modifications. In oneembodiment, a modified antibody, or antigen-binding fragment thereof,contains six modifications. In one embodiment, a modified antibody, orantigen-binding fragment thereof, contains seven modifications. In oneembodiment, a modified antibody, or antigen-binding fragment thereof,contains eight modifications. In one embodiment, a modified antibody, orantigen-binding fragment thereof, contains nine modifications. In oneembodiment, a modified antibody, or antigen-binding fragment thereof,contains ten modifications. In one embodiment, a modified antibody, orantigen-binding fragment thereof, contains up to twenty modifications.

Provided herein is an antibody, or antigen-binding fragment thereof,comprising a light chain variable region having an amino acid sequenceset forth as SEQ ID NO: 93 (VK1AA) and a heavy chain variable regionhaving an amino acid sequence set forth as SEQ ID NO: 89 (VH1A2).

Provided herein is a humanized and deimmunized antibody, orantigen-binding fragment thereof, comprising a heavy chain variableregion having an amino acid sequence set forth as any one of SEQ ID NOS:88, 89, 90, 91 and 92.

Provided herein is a humanized and deimmunized antibody, orantigen-binding fragment thereof, comprising a light chain variableregion having an amino acid sequence set forth as any one of SEQ ID NOS:93, 94, 95, 96, 97, 100, 102, and 103.

Provided herein is a humanized and deimmunized antibody, orantigen-binding fragment thereof, that binds endoglin, comprising aheavy chain variable region having an amino acid sequence set forth asSEQ ID NO: 89 and a light chain variable region having an amino acidsequence set forth as SEQ ID NO: 93, wherein:

(i) the heavy chain variable region further comprises one or moremodifications selected from the group consisting of a substitution ofglycine (G) by alanine (A) or serine (S) at position 49; a substitutionof alanine (A) by isoleucine (I) at position 51; a substitution oflysine (K) by arginine (R) or asparagine (Q) at position 52b; asubstitution of leucine (L) by valine (V) at position 78 utilizing theKabat numbering system; and

(ii) the light chain variable region further comprises one or moremodifications selected from the group consisting of a substitution ofmethionine (M) by leucine (L) at position 4; a substitution of alanine(A) by valine (V) at position 19; a substitution of threonine (T) byserine (S) at position 22; a substitution of alanine (A) by isoleucine(I) at position 48; and a substitution of threonine (T) by serine (S) atposition 51 utilizing the Kabat numbering system

Provided herein is a humanized and deimmunized antibody, orantigen-binding fragment thereof comprising a heavy chain variableregion having an amino acid sequence set forth as SEQ ID NO: 88, 89, 90,91 and 92; and a light chain variable region having an amino acidsequence set forth as SEQ ID NO: 93, 95, 96, 97, 100, 102, or 103.

In addition to the aforementioned examples and embodiments, a modified ahumanized and deimmunized antibody, or antigen-binding fragment thereof,with one or more amino acid modifications in one or more T-cell epitopesare contemplated herein. In one non-limiting example, provided hereinare antibodies, or antigen-binding fragments thereof, having at leastone modification in at least one T-cell epitope. In another non-limitingexample, provided herein are antibodies, or antigen-binding fragmentsthereof, having at least one amino acid modification in 1, 2, 3, 4, 5,6, or 7 of the T-cell epitopes described herein. Additional non-limitingexamples include antibodies, or antigen-binding fragments thereof,having more than one amino acid modification in more than one T-cellepitope. Any combination of the amino acid modifications in any numberof the antibodies, or antigen-binding fragments thereof, T-cell epitopesdescribed above are contemplated herein.

T-Cell Epitopes and Allotype Frequency

Individual epitopes found within antigens can be preferentiallypresented by specific MHC class II allotypes, and similarly otherspecific epitopes within the same antigen may not be presented on MHCclass II molecules at all. Such associations of particular epitopes withspecific MHC class II molecules have been shown to depend upon the MHCclass II allotype of the individual. The association of a specificepitope with a specific allotype can also be considered when modifyingantibodies, or antigen-binding fragments thereof, for the removal ofT-cell epitopes. Such considerations can allow for the highly specificmodification of an antibody, or antigen-binding fragment thereof, forspecific allotypes (e.g., for specific populations of subjects havingcertain MHC class II allotypes). The MHC class II allotype of a subjector subjects can be easily determined by genotyping methods known in theart, and the association of T-cell epitopes with the given allotype thuseasily identified, for consideration in modification of antibodies, orantigen-binding fragments thereof, tailored to that allotype.Identification of associations between T-cell epitopes and MHC class IIallotypes are described in more detail in the examples below.Contemplated herein are modified antibodies, or antigen-bindingfragments thereof, that have T-cell epitope modifications tailored tothe MHC class II associations identified for the given epitopes.

D. Anti-Endoglin Antibodies

Simultaneous incorporation of all of the FR and/or CDR encoding nucleicacids and all of the selected amino acid position changes can beaccomplished by a variety of methods known to those skilled in the art,including for example, recombinant and chemical synthesis. For example,simultaneous incorporation can be accomplished by, for example,chemically synthesizing the nucleotide sequence for the acceptorvariable region, fused together with the donor CDR encoding nucleicacids, and incorporating at the positions selected for harboringvariable amino acid residues a plurality of corresponding amino acidcodons.

One can recognize that the a humanized and deimmunized antibodies andantigen-binding fragments thereof generated using the methods describedherein can be tested using the assays provided herein or known in theart for the ability to bind to endoglin using conventional methodsincluding, but not limited to, ELISA. Affinity of antibodies describedherein can also be determined using conventional methods including, butnot limited to, Biacore or surface plasmon resonance. The activity ofthe antibodies may tested using a competitive assay such as described inthe Examples, to determine if the antibodies inhibit BMP9 function. Cellsignaling pathways to assess the activity of, for example Smad 1/5/8 areknown in the art and are contemplated for use herein.

The antibodies and antigen binding fragments thereof described hereinwere constructed by humanization of the V_(H) and V_(L) sequences of theTRC105 antibody. To accomplish this humanization, a 3-dimensional modelof the V_(H) and V_(L) chains of TRC105 was created and analyzed. TheV_(H) and V_(L) sequences were then compared individually to a databaseof human germline sequences, from which human V_(H) and V_(L) sequenceswere chosen based on their homology to the V_(H) and V_(L) sequences ofTRC105. The human V_(L) sequence chosen for humanization was O2/O12(VK1-39) (SEQ ID NO. 2). O2/O12 has a sequence identity with TRC105 of65% and the gene is highly expressed in the human germline repertoire.The human V_(H) sequence chosen for humanization was VH3-15 (SEQ ID NO.40). VH3-15 has sequence identity with TRC105 of 70% and is expressedwith reasonable frequency in the human germline repertoire. The aminoacid positions which were different between TRC105 and the humansequences were examined in the 3D model of TRC105 to determine whichsubstitutions would be considered for modification. Amino acid selectioncriteria based on the 3D model analysis included, but was not limitedto, for example, steric effects related to the amino acid, relativecharge of the amino acid, and the location of the amino acid within thevariable heavy and/or light chains. The identified and proposedsubstitutions for the human framework regions are incorporated into theO2 and VH3-15 human framework regions, and the CDRs of TRC105 aregrafted into the corresponding O2 and VH3-15 human framework regionsresulting in a multitude of humanized antibodies or antigen-bindingfragments. Additionally, the FR-4 of the light chain is derived fromhuman J germline sequence Jk4. Similarly, the FR-4 of the heavy chain isderived from human J germline sequence JH4.

Antibodies and antigen-binding fragments thereof can have a variableheavy (V_(H)) chain, a variable light (V_(L)) chain, both, or bindingportions thereof. In one embodiment, the V_(H) chain has an amino acidsequence set forth as any of SEQ ID NOS: 41-43, or a binding portionthereof. Such V_(H) chains can have framework regions sequences setforth as any of SEQ ID NOS: 44-62. In another embodiment, the V_(L)chain has an amino acid sequence set forth as any of SEQ ID NOS: 3-5, ora binding portion thereof. Such V_(L) chains can have framework regionssequences set forth as any of SEQ ID NOS: 6-38.

Provided herein is an antibody, or antigen-binding fragment thereof,comprising a light chain variable region having an amino acid sequenceset forth as SEQ ID NO: 3 and a heavy chain variable region having anamino acid sequence set forth as SEQ ID NO: 41.

Provided herein is an antibody, or antigen-binding fragment thereof,comprising a light chain variable region having an amino acid sequenceset forth as SEQ ID NO: 3 and a heavy chain variable region having anamino acid sequence set forth as SEQ ID NO: 41, wherein: the heavy chainvariable region further comprises one or more modifications selectedfrom the group consisting of a substitution of glycine (G) by alanine(A) at position 49; a substitution of asparagine (N) by serine (S) atposition 76; a substitution of threonine (T) by arginine (R) at position77; a substitution of leucine (L) by valine (V) at position 78; asubstitution of asparagine (N) by isoleucine (I) at position 82a; asubstitution of valine (V) by isoleucine (I) or leucine (L) at position89; a substitution of threonine (T) by arginine (R) or glycine (G) atposition 94; a substitution of leucine (L) by threonine (T) at position108; a substitution of valine (V) by leucine (L) at position 109; and asubstitution of serine (S) by alanine (A) at position 113 utilizing theKabat numbering system; and the light chain variable region furthercomprises one or more modifications selected from the group consistingof a substitution of aspartic acid (D) by glutamine (Q) at position 1; asubstitution of glutamine (Q) by valine (V) at position 3; asubstitution of methionine (M) by leucine (L) at position 4; asubstitution of threonine (T) by serine (S) at position 5; asubstitution of tyrosine (Y) by phenylalanine (F) at position 36; asubstitution of leucine (L) by proline (P) at position 46; asubstitution of leucine (L) by tryptophan (W) at position 47; asubstitution of serine (S) by valine (V) or alanine (A) at position 60;a substitution of aspartic acid (D) by serine (S) at position 70; asubstitution of phenylalanine (F) by tyrosine (Y) at position 71; asubstitution of glutamine (G) by alanine (A) at position 100; and asubstitution of isoleucine (I) by leucine (L) at position 106 utilizingthe Kabat numbering system.

Provided herein is an antibody, or antigen-binding fragment thereof,that binds endoglin comprising a heavy chain variable region having anamino acid sequence set forth as SEQ ID NO: 41, 42, or 43; and a lightchain variable region having an amino acid sequence set forth as SEQ IDNO: 3, 4, or 5. An antibody, or antigen-binding fragment thereof, cancomprise a heavy chain variable region having an amino acid sequence setforth as SEQ ID NO: 41 and a light chain variable region having an aminoacid sequence set forth as SEQ ID NO: 3. An antibody, or antigen-bindingfragment thereof, can comprise a heavy chain variable region having anamino acid sequence set forth as SEQ ID NO: 41 and a light chainvariable region having an amino acid sequence set forth as SEQ ID NO: 4.An antibody, or antigen-binding fragment thereof, can comprise a heavychain variable region having an amino acid sequence set forth as SEQ IDNO: 41 and a light chain variable region having an amino acid sequenceset forth as SEQ ID NO: 5. An antibody, or antigen-binding fragmentthereof, can comprise a heavy chain variable region having an amino acidsequence set forth as SEQ ID NO: 42 and a light chain variable regionhaving an amino acid sequence set forth as SEQ ID NO: 3. An antibody, orantigen-binding fragment thereof, can comprise a heavy chain variableregion having an amino acid sequence set forth as SEQ ID NO: 42 and alight chain variable region having an amino acid sequence set forth asSEQ ID NO: 4. An antibody, or antigen-binding fragment thereof, cancomprise a heavy chain variable region having an amino acid sequence setforth as SEQ ID NO: 42 and a light chain variable region having an aminoacid sequence set forth as SEQ ID NO: 5. An antibody, or antigen-bindingfragment thereof, can comprise a heavy chain variable region having anamino acid sequence set forth as SEQ ID NO: 43 and a light chainvariable region having an amino acid sequence set forth as SEQ ID NO: 3.An antibody, or antigen-binding fragment thereof, can comprise a heavychain variable region having an amino acid sequence set forth as SEQ IDNO: 43 and a light chain variable region having an amino acid sequenceset forth as SEQ ID NO: 4. An antibody, or antigen-binding fragmentthereof, can comprise a heavy chain variable region having an amino acidsequence set forth as SEQ ID NO: 43 and a light chain variable regionhaving an amino acid sequence set forth as SEQ ID NO: 5.

In any of such embodiments, a heavy chain variable region can furthercomprise one or more modifications selected from the group consistingof: a substitution of asparagine (N) by serine (S) at position 76; asubstitution of threonine (T) by arginine (R) at position 77; asubstitution of asparagine (N) by isoleucine (I) at position 82a; asubstitution of valine (V) by isoleucine (I) or leucine (L) at position89; a substitution of threonine (T) by glycine (G) at position 94; asubstitution of leucine (L) by threonine (T) at position 108; asubstitution of valine (V) by leucine (L) at position 109; and asubstitution of serine (S) by alanine (A) a position 113; and the lightchain variable region can further comprise one or more modificationsselected from the group consisting of: a substitution of aspartic acid(D) by glutamine (Q) at position 1; a substitution of glutamine (Q) byvaline (V) at position 3; a substitution of threonine (T) by serine (S)at position 5; a substitution of tyrosine (Y) by phenylalanine (F) aposition 36; a substitution of serine (S) by valine (V) or alanine (A)at position 60; a substitution of aspartic acid (D) by serine (S) atposition 70; a substitution of glycine (G) by alanine (A) at position100, and a substitution of isoleucine (I) by leucine (L) at position 106utilizing the Kabat numbering system.

Provided herein is an antibody, or antigen-binding fragment thereof,that binds endoglin, comprising a heavy chain variable region and alight chain variable region, wherein said heavy chain variable regioncomprises:

(i) a CDR1 of SEQ ID NO: 66, a CDR2 of SEQ ID NO: 67, and a CDR3 of SEQID NO: 68;

(ii) a heavy chain FR1 having the amino acid sequence of SEQ ID NO: 44or the amino acid sequence of SEQ ID NO: 44 except for one or moreconservative substitutions;

(iii) a heavy chain FR2 having the amino acid sequence of SEQ ID NO: 45or the amino acid sequence of SEQ ID NO: 45 except for a substitution ofglycine (G) by alanine (A) at position 49 utilizing the Kabat numberingsystem; and

(iv) a heavy chain FR3 having the amino acid sequence of SEQ ID NO: 47or the amino acid sequence of SEQ ID NO: 47 except for one or moresubstitutions selected from the group consisting of:

-   -   (a) a substitution of asparagine (N) by serine (S) at position        76;    -   (b) a substitution of threonine (T) by arginine (R) at position        77;    -   (c) a substitution of leucine (L) by valine (V) at position 78;    -   (d) a substitution of asparagine (N) by isoleucine (I) at        position 82a;    -   (e) a substitution of valine (V) by isoleucine (I) or        leucine (L) at position 89; and    -   (f) a substitution of threonine (T) by arginine (R) or        glycine (G) at position 94 utilizing the Kabat numbering system;        and

(v) a heavy chain FR4 having the amino acid sequence of SEQ ID NO: 56 orthe amino acid sequence of SEQ ID NO: 56 except for one or moresubstitutions selected from the group consisting of:

-   -   (a) a substitution of leucine (L) by threonine (T) at position        108;    -   (b) a substitution of valine (V) by leucine (L) at position 109;        and    -   (c) a substitution of serine (S) by alanine (A) at position 113        utilizing the Kabat numbering system;

and said light chain variable region comprises:

(i) a CDR1 of SEQ ID NO: 63, a CDR2 of SEQ ID NO: 64, and a CDR3 of SEQID NO: 65;

(ii) a light chain FR1 having the amino acid sequence of SEQ ID NO: 6 orthe amino acid sequence of SEQ ID NO: 6 except for one or moresubstitutions selected from the group consisting of:

-   -   (a) a substitution of aspartic acid (D) by glutamine (Q) at        position 1;    -   (b) a substitution of glutamine (Q) by valine (V) at position 3;    -   (c) a substitution of methionine (M) by leucine (L) at position        4; and    -   (d) a substitution of threonine (T) by serine (S) at position 5;        utilizing the Kabat numbering system; and

(iii) a light chain FR2 having the amino acid sequence of SEQ ID NO: 20or the amino acid sequence of SEQ ID NO: 20 except for one or moresubstitutions selected from the group consisting of:

-   -   (a) a substitution of tyrosine (Y) by phenylalanine (F) at        position 36;    -   (b) a substitution of leucine (L) by proline (P) at position 46;        and    -   (c) a substitution of leucine (L) by tryptophan (W) at position        47 utilizing the Kabat numbering system; and

(iv) a light chain FR3 having the amino acid sequence of SEQ ID NO: 28or the amino acid sequence of SEQ ID NO: 28 except for one or moresubstitutions selected from the group consisting of:

-   -   (a) a substitution of serine (S) by valine (V) or alanine (A) at        position 60;    -   (b) a substitution of aspartic acid (D) by serine (S) at        position 70; and    -   (b) a substitution of phenylalanine (F) by tyrosine (Y) at        position 71 utilizing the Kabat numbering system; and

(v) a light chain FR4 having the amino acid sequence of SEQ ID NO: 35 orthe amino acid sequence of SEQ ID NO: 35 except for one or moresubstitutions selected from the group consisting of:

-   -   (a) a substitution of glycine (G) by alanine (A) at position        100; and    -   (b) a substitution of isoleucine (I) by leucine (L) at position        106 utilizing the Kabat numbering system.

An antibody, or antigen-binding fragment thereof, provided herein cancomprise a heavy chain variable region CDR1 having an amino acidsequence as set forth in SEQ ID NO: 66, a heavy chain variable regionCDR2 having an amino acid sequence as set forth in SEQ ID NO: 67, aheavy chain variable region CDR3 having an amino acid sequence as setforth in SEQ ID NO: 68, a light chain variable region CDR1 having anamino acid sequence as set forth in SEQ ID NO: 63, a light chainvariable region CDR2 having an amino acid sequence as set forth in SEQID NO: 64, and a light chain variable region CDR3 having an amino acidsequence as set forth in SEQ ID NO: 65.

In one embodiment, the antibody, or antigen-binding fragment thereofbinds endoglin and comprises a heavy chain variable region FR1 having anamino acid sequence as set forth in SEQ ID NO: 44; a heavy chainvariable region FR2 having an amino acid sequence as set forth in SEQ IDNO: 45; a heavy chain variable region FR3 having an amino acid sequenceas set forth in SEQ ID NO: 47; a heavy chain variable region FR4 havingan amino acid sequence as set forth in SEQ ID NO: 56.

In another embodiment, the antibody, or antigen-binding fragment thereofbinds endoglin and comprises a heavy chain variable region FR1 having anamino acid sequence as set forth in SEQ ID NO: 44; a heavy chainvariable region FR2 having an amino acid sequence as set forth in SEQ IDNO: 46; a heavy chain variable region FR3 having an amino acid sequenceas set forth in SEQ ID NO: 48; a heavy chain variable region FR4 havingan amino acid sequence as set forth in SEQ ID NO: 56.

In another embodiment, the antibody, or antigen-binding fragmentthereof, comprises a light chain variable region FR1 having an aminoacid sequence as set forth in SEQ ID NO: 6; a light chain variableregion FR2 having an amino acid sequence as set forth in SEQ ID NO: 20;a light chain variable region FR3 having an amino acid sequence as setforth in SEQ ID NO: 28; and a light chain variable region FR4 having anamino acid sequence as set forth in SEQ ID NO: 35.

In another embodiment, the antibody, or antigen-binding fragment thereofbinds endoglin and comprises a light chain variable region FR1 having anamino acid sequence as set forth in SEQ ID NO: 6; a light chain variableregion FR2 having an amino acid sequence as set forth in SEQ ID NO: 21;a light chain variable region FR3 having an amino acid sequence as setforth in SEQ ID NO: 29; and a light chain variable region FR4 having anamino acid sequence as set forth in SEQ ID NO: 35.

In another embodiment, the antibody, or antigen-binding fragment thereofbinds endoglin and comprises a light chain variable region FR1 having anamino acid sequence as set forth in SEQ ID NO: 7; a light chain variableregion FR2 having an amino acid sequence as set forth in SEQ ID NO: 21;a light chain variable region FR3 having an amino acid sequence as setforth in SEQ ID NO: 29; and a light chain variable region FR4 having anamino acid sequence as set forth in SEQ ID NO: 35.

Provided herein is an antibody, or antigen-binding fragment thereof,comprising a heavy chain variable region having an amino acid sequenceset forth as SEQ ID NO: 42 and a light chain variable region having anamino acid sequence set forth as SEQ ID NO: 4.

Provided herein is an antibody, or antigen-binding fragment thereof,that binds endoglin, comprising a light chain variable region having anamino acid sequence set forth as SEQ ID NO: 4 and a heavy chain variableregion having an amino acid sequence set forth as SEQ ID NO: 42,wherein: said heavy chain variable region further comprises one or moremodifications selected from the group consisting of a substitution ofglycine (G) by alanine (A) at position 49; a substitution of asparagine(N) by serine (S) at position 76; a substitution of threonine (T) byarginine (R) at position 77; a substitution of leucine (L) by valine (V)at position 78; a substitution of asparagine (N) by isoleucine (I) atposition 82a; a substitution of valine (V) by isoleucine (I) or leucine(L) at position 89; a substitution of arginine (R) by threonine (T) orglycine (G) at position 94; a substitution of leucine (L) by threonine(T) at position 108; a substitution of valine (V) by leucine (L) atposition 109; and a substitution of serine (S) by alanine (A) atposition 113 utilizing the Kabat numbering system; and the light chainvariable region further comprises one or more modifications selectedfrom the group consisting of a substitution of aspartic acid (D) byglutamine (Q) at position 1; a substitution of glutamine (Q) by valine(V) at position 3; a substitution of methionine (M) by leucine (L) atposition 4; a substitution of threonine (T) by serine (S) at position 5;a substitution of tyrosine (Y) by phenylalanine (F) at position 36; asubstitution of proline (P) by leucine (L) at position 46; asubstitution of tryptophan (W) by leucine (L) at position 47; asubstitution of serine (S) by valine (V) or alanine (A) at position 60;a substitution of aspartic acid (D) by serine (S) at position 70; asubstitution of tyrosine (Y) by phenylalanine (F) at position 71; asubstitution of glutamine (G) by alanine (A) at position 100; and asubstitution of isoleucine (I) by leucine (L) at position 106 utilizingthe Kabat numbering system

Provided herein is an antibody, or antigen-binding fragment thereof,that binds endoglin, comprising a heavy chain variable region and alight chain variable region,

wherein said heavy chain variable region comprises:

(i) a CDR1 of SEQ ID NO: 66, a CDR2 of SEQ ID NO: 67, and a CDR3 of SEQID NO: 68;

(ii) a heavy chain FR1 having the amino acid sequence of SEQ ID NO: 44or the amino acid sequence of SEQ ID NO: 44 except for one or moreconservative substitutions;

(iii) a heavy chain FR2 having the amino acid sequence of SEQ ID NO: 45or the amino acid sequence of SEQ ID NO: 45 except for a substitution ofglycine (G) by alanine (A) at position 49 utilizing the Kabat numberingsystem; and

(iv) a heavy chain FR3 having the amino acid sequence of SEQ ID NO: 47or the amino acid sequence of SEQ ID NO: 47 except for one or moresubstitutions selected from the group consisting of:

-   -   (a) a substitution of asparagine (N) by serine (S) at position        76;    -   (b) a substitution of threonine (T) by arginine (R) at position        77;    -   (c) a substitution of leucine (L) by valine (V) at position 78;    -   (d) a substitution of asparagine (N) by isoleucine (I) at        position 82a;    -   (e) a substitution of valine (V) by isoleucine (I) or        leucine (L) at position 89; and    -   (f) a substitution of arginine (R) by threonine (T) or        glycine (G) at position 94 utilizing the Kabat numbering system;        and

(v) a heavy chain FR4 having the amino acid sequence of SEQ ID NO: 56 orthe amino acid sequence of SEQ ID NO: 56 except for one or moresubstitutions selected from the group consisting of:

-   -   (a) a substitution of leucine (L) by threonine (T) at position        108;    -   (b) a substitution of valine (V) by leucine (L) at position 109;        and    -   (c) a substitution of serine (S) by alanine (A) at position 113        utilizing the Kabat numbering system;

and said light chain variable region comprises:

(i) a CDR1 of SEQ ID NO: 63, a CDR2 of SEQ ID NO: 64, and a CDR3 of SEQID NO: 65;

(ii) a light chain FR1 having the amino acid sequence of SEQ ID NO: 6 orthe amino acid sequence of SEQ ID NO: 6 except for one or moresubstitutions selected from the group consisting of:

-   -   (a) a substitution of aspartic acid (D) by glutamine (Q) at        position 1;    -   (b) a substitution of glutamine (Q) by valine (V) at position 3;    -   (c) a substitution of methionine (M) by leucine (L) at position        4; and    -   (d) a substitution of threonine (T) by serine (S) at position 5;        utilizing the Kabat numbering system; and

(iii) a light chain FR2 having the amino acid sequence of SEQ ID NO: 21or the amino acid sequence of SEQ ID NO: 20 except for one or moresubstitutions selected from the group consisting of:

-   -   (a) a substitution of tyrosine (Y) by phenylalanine (F) at        position 36;    -   (b) a substitution of proline (P) by leucine (L) at position 46;        and    -   (c) a substitution of tryptophan (W) by leucine (L) at position        47 utilizing the Kabat numbering system; and

(iv) a light chain FR3 having the amino acid sequence of SEQ ID NO: 29or the amino acid sequence of SEQ ID NO: 28 except for one or moresubstitutions selected from the group consisting of:

-   -   (a) a substitution of serine (S) by valine (V) or alanine (A) at        position 60;    -   (b) a substitution of aspartic acid (D) by serine (S) at        position 70; and    -   (b) a substitution of tyrosine (Y) by phenylalanine (F) at        position 71 utilizing the Kabat numbering system; and

(v) a light chain FR4 having the amino acid sequence of SEQ ID NO: 35 orthe amino acid sequence of SEQ ID NO: 35 except for one or moresubstitutions selected from the group consisting of:

-   -   (a) a substitution of glycine (G) by alanine (A) at position        100; and    -   (b) a substitution of isoleucine (I) by leucine (L) at position        106 utilizing the Kabat numbering system.

A substantial portion of a variable domain will include three CDRregions, together with their intervening framework regions. The portioncan also include at least about 50% of either or both of the first andfourth framework regions, the 50% being the C-terminal 50% of the firstframework region and the N-terminal 50% of the fourth framework region.Additional residues at the N-terminal or C-terminal end of thesubstantial part of the variable domain may be those not normallyassociated with naturally occurring variable domain regions. Forexample, construction of humanized endoglin antibodies andantigen-binding fragments described herein made by recombinant DNAtechniques can result in the introduction of N- or C-terminal residuesencoded by linkers introduced to facilitate cloning or othermanipulation steps. Other manipulation steps include the introduction oflinkers to join variable domains to further protein sequences includingimmunoglobulin heavy chains, other variable domains (for example in theproduction of diabodies) or protein labels as discussed in more detailbelow.

Humanized endoglin CDR3 regions having amino acid sequencessubstantially as set out as the CDR3 regions of the antibodies describedherein will be carried in a structure which allows for binding of theCDR3 regions to endoglin. The structure for carrying the CDR3s can be ofan antibody heavy or light chain sequence or substantial portion thereofin which the CDR3 regions are located at locations corresponding to theCDR3 region of naturally-occurring V_(H) and V_(L) antibody variabledomains encoded by rearranged immunoglobulin genes.

In one non-limiting example, provided herein are antibodies or antigenbinding fragments thereof containing a variable heavy chain having aCDR3 which has an amino acid sequence set forth as SEQ ID NO: 68 and/ora variable light chain having a CDR3 which has an amino acid sequenceset forth as SEQ ID NO: 65. In one embodiment, the variable heavy chainhas an amino acid sequence set forth as SEQ ID NO: 40 except for asubstitution of the CDR3 by the CDR3 amino sequence set forth as SEQ IDNO: 68. In another embodiment, the variable light chain has an aminoacid sequence set forth as SEQ ID NO: 2 except for a substitution of theCDR3 by the CDR3 amino acid sequence set forth as SEQ ID NO: 65.Additionally, such CDR3 containing variable regions/chains can compriseone or more FR amino acid sequences set forth as, for example, describedabove (or such FRs containing one or more additional modifications),where the antibodies or antigen binding fragments have 3 CDRs and 4 FRsin each of the VH and VL regions, have specific binding activity forendoglin. Additionally, various antibody J segments can also besubstituted within these variable regions for further variation withinthe variable region chains.

TABLE 1 In one aspect, variable heavy and light chains described hereincan also be created by further replacing FR4 sequences. In oneembodiment, heavy chain FR4 sequences can be substituted for one of thefollowing: SEQ ID NO: Kabat-Number 103 104 105 106 107 108 109 110 111112 113 76 FRM4 from JH1, W G Q G T L V T V S S JH4 or JH5 77 FRM4 fromJH2 W G R G T L V T V S S 78 FRM4 from JH3 W G Q G T M V T V S S 79 FRM4from JH6 W G Q G T T V T V S S

TABLE 2 In one embodiment, light chain FR4 sequences can be substitutedfor one of the following: SEQ ID NO: Kabat Number 98 99 100 101 102 103104 105 106 107 80 JK1 F G Q G T K V E I K 81 JK2 F G Q G T K L E I K 82JK3 F G P G T K V D I K 83 JK4 F G G G T K V E I K 84 JK5 F G Q G T R LE I K

Further provided herein are humanized versions of anti-endoglinantibodies alternatively named “superhumanized” anti-endoglin antibodiesor antigen-binding fragments thereof. Such superhumanized antibodies, orantigen-binding fragments thereof, can comprise a light chain variableregion having an amino acid sequence set forth as SEQ ID NOS: 71 or 72and a heavy chain variable region having an amino acid sequence setforth as SEQ ID NO: 75.

In another aspect, the present application provides a humanized antibodycapable of competing with a humanized anti-endoglin antibody orantigen-binding described herein under conditions in which at least 5%of an antibody having the V_(H) and V_(L) sequences of the antibody isblocked from binding to endoglin by competition with such an antibody inan ELISA assay.

Provided herein are neutralizing antibodies or antigen-binding fragmentsthat bind to endoglin and modulate the activity of endoglin or inhibitbinding of BMP9 to a TGF beta receptor.

In one aspect, the antigen-binding fragment of any one of the humanizedantibodies described above is a Fab, a Fab′, a Fd, a F(ab′)₂, a Fv, ascFv, a single chain binding polypeptide (e.g., a scFv with Fc portion)or any other functional fragment thereof as described herein.

Antibodies or antigen-binding fragments described herein are useful indetection or diagnostic applications as described in more detail below.

Antibodies, or antigen-binding fragments thereof, described herein canbe further modified to alter the specific properties of the antibodywhile retaining the desired functionality, if needed. For example, inone embodiment, the compound can be modified to alter a pharmacokineticproperty of the compound, such as in vivo stability, solubility,bioavailability or half-life. Antibodies, or antigen-binding fragmentsthereof, described herein can further comprise a therapeutic moiety, adetectable moiety, or both, for use in diagnostic and/or therapeuticapplications.

Antibodies, or antigen-binding fragments thereof, described herein canalso be used as immunoconjugates. As used herein, for purposes of thespecification and claims, immunoconjugates refer to conjugates comprisedof the humanized anti-endoglin antibodies or fragments thereof accordingto the present invention and at least one therapeutic label. Suchantitumor agents are known in the art and include, but not limited to,toxins, drugs, enzymes, cytokines, radionuclides, and photodynamicagents. Toxins include, but are not limited to, ricin A chain, mutantPseudomonas exotoxins, diphtheria toxoid, streptonigrin, boamycin,saporin, gelonin, and pokeweed antiviral protein. Drugs includedaunorubicin, methotrexate, and calicheamicins. Radionuclides includeradiometals. Cytokines include, but are not limited to, transforminggrowth factor (TGF)-β, interleukins, interferons, and tumor necrosisfactors. Photodynamic agents include, but are not limited to, porphyrinsand their derivatives. Additional therapeutic labels will be known inthe art and are also contemplated herein. The methods for complexing theanti-endoglin mAbs or a fragment thereof with at least one antitumoragent are well known to those skilled in the art (i.e., antibodyconjugates as reviewed by Ghetie et al., 1994, Pharmacol. Ther.63:209-34). Such methods may utilize one of several availableheterobifunctional reagents used for coupling or linking molecules.Additional radionuclides are further described herein along withadditional methods for linking molecules, such as therapeutic labels.

Antibodies, or antigen-binding fragments thereof, can be modified usingtechniques known in the art for various purposes such as, for example,by addition of polyethylene glycol (PEG). PEG modification (PEGylation)can lead to one or more of improved circulation time, improvedsolubility, improved resistance to proteolysis, reduced antigenicity andimmunogenicity, improved bioavailability, reduced toxicity, improvedstability, and easier formulation (for a review see, Francis et al.,International Journal of Hematology 68:1-18, 1998).

In the case of an antigen-binding fragment which does not contain an Fcportion, an Fc portion can be added to (e.g., recombinantly) thefragment, for example, to increase half-life of the antigen-bindingfragment in circulation in blood when administered to a patient. Choiceof an appropriate Fc region and methods of to incorporate such fragmentsare known in the art. Incorporating a Fc region of an IgG into apolypeptide of interest so as to increase its circulatory half-life, butso as not to lose its biological activity can be accomplished usingconventional techniques known in the art such as, for example, describedin U.S. Pat. No. 6,096,871, which is hereby incorporated by reference inits entirety. Fc portions of antibodies can be further modified toincrease half-life of the antigen-binding fragment in circulation inblood when administered to a patient. Modifications can be determinedusing conventional means in the art such as, for example, described inU.S. Pat. No. 7,217,798, which is hereby incorporated by reference inits entirety.

Other methods of improving the half-life of antibody-based fusionproteins in circulation are also known such as, for example, describedin U.S. Pat. Nos. 7,091,321 and 6,737,056, each of which is herebyincorporated by reference. Additionally, antibodies and antigen-bindingfragments thereof may be produced or expressed so that they do notcontain fucose on their complex N-glycoside-linked sugar chains. Theremoval of the fucose from the complex N-glycoside-linked sugar chainsis known to increase effector functions of the antibodies andantigen-binding fragments, including but not limited to, antibodydependent cell-mediated cytotoxicity (ADCC) and complement dependentcytotoxicity (CDC). Similarly, antibodies or antigen-binding fragmentsthereof that can bind endoglin can be attached at their C-terminal endto all or part of an immunoglobulin heavy chain derived from anyantibody isotype, e.g., IgG, IgA, IgE, IgD and IgM and any of theisotype sub-classes, particularly IgG1, IgG2b, IgG2a, IgG3 and IgG4.

Additionally, the antibodies or antigen-binding fragments describedherein can also be modified so that they are able to cross theblood-brain barrier. Such modification of the antibodies orantigen-binding fragments described herein allows for the treatment ofbrain diseases such as glioblastoma multiforme (GBM). Exemplarymodifications to allow proteins such as antibodies or antigen-bindingfragments to cross the blood-brain barrier are described in US PatentApplication Publication 2007/0082380 which is hereby incorporated byreference in its entirety.

Glycosylation of immunoglobulins has been shown to have significanteffects on their effector functions, structural stability, and rate ofsecretion from antibody-producing cells (Leatherbarrow et al., Mol.Immunol. 22:407 (1985)). The carbohydrate groups responsible for theseproperties are generally attached to the constant (C) regions of theantibodies. For example, glycosylation of IgG at asparagine 297 in theC_(H)2 domain is required for full capacity of IgG to activate theclassical pathway of complement-dependent cytolysis (Tao and Morrison,J. Immunol. 143:2595 (1989)). Glycosylation of IgM at asparagine 402 inthe C_(H)3 domain is necessary for proper assembly and cytolyticactivity of the antibody (Muraoka and Shulman, J. Immunol. 142:695(1989)). Removal of glycosylation sites as positions 162 and 419 in theC_(H)1 and C_(H)3 domains of an IgA antibody led to intracellulardegradation and at least 90% inhibition of secretion (Taylor and Wall,Mol. Cell. Biol. 8:4197 (1988)). Additionally, antibodies andantigen-binding fragments thereof may be produced or expressed so thatthey do not contain fucose on their complex N-glycoside-linked sugarchains. The removal of the fucose from the complex N-glycoside-linkedsugar chains is known to increase effector functions of the antibodiesand antigen-binding fragments, including but not limited to, antibodydependent cell-mediated cytotoxicity (ADCC) and complement dependentcytotoxicity (CDC). These “defucosylated” antibodies and antigen-bindingfragments may be produced through a variety of systems utilizingmolecular cloning techniques known in the art, including but not limitedto, transgenic animals, transgenic plants, or cell-lines that have beengenetically engineered so that they no longer contain the enzymes andbiochemical pathways necessary for the inclusion of a fucose in thecomplex N-glycoside-linked sugar chains (also known asfucosyltransferase knock-out animals, plants, or cells). Non-limitingexamples of cells that can be engineered to be fucosyltransferaseknock-out cells include CHO cells, SP2/0 cells, NS0 cells, and YB2/0cells.

Glycosylation of immunoglobulins in the variable (V) region has alsobeen observed. Sox and Hood reported that about 20% of human antibodiesare glycosylated in the V region (Proc. Natl. Acad. Sci. USA 66:975(1970)). Glycosylation of the V domain is believed to arise fromfortuitous occurrences of the N-linked glycosylation signalAsn-Xaa-Ser/Thr in the V region sequence and has not been recognized inthe art as playing a role in immunoglobulin function.

Glycosylation at a variable domain framework residue can alter thebinding interaction of the antibody with antigen. The present inventionincludes criteria by which a limited number of amino acids in theframework or CDRs of a humanized immunoglobulin chain are chosen to bemutated (e.g., by substitution, deletion, or addition of residues) inorder to increase the affinity of an antibody.

Affinity for binding a pre-determined polypeptide antigen can,generally, be modulated by introducing one or more mutations into the Vregion framework, typically in areas adjacent to one or more CDRs and/orin one or more framework regions. Typically, such mutations involve theintroduction of conservative amino acid substitutions that eitherdestroy or create the glycosylation site sequences but do notsubstantially affect the hydropathic structural properties of thepolypeptide. Typically, mutations that introduce a proline residue areavoided. Glycosylation of antibodies and antigen-binding fragmentsthereof is further described in U.S. Pat. No. 6,350,861, which isincorporated by reference herein with respect to glycosylation.

Antibodies, or antigen-binding fragments thereof, can be formulated forshort-term delivery or extended (long term) delivery.

Antibodies, or antigen-binding fragments thereof, that bind to endoglincan also be used for purification of endoglin and/or to detect endoglinlevels in a sample or patient to detect or diagnose a disease ordisorder associated with endoglin as described in more detail below.

Humanized antibodies, antigen-binding fragments, and binding proteinswhich bind endoglin generated using such methods can be tested for oneor more of their binding affinity, avidity, and neutralizingcapabilities. Useful humanized antibodies, antigen-binding fragments,and binding proteins can be used to administer a patient to prevent,inhibit, manage or treat a fibrotic condition disease or disorder.

Provided herein are methods of identifying humanized antibodies orantigen-binding fragments thereof that bind to endoglin. Antibodies andantigen-binding fragments can be evaluated for one or more of bindingaffinity, association rates, disassociation rates and avidity. In oneaspect, antibodies can be evaluated for their ability to neutralize theactivity of endoglin or a polypeptide in which the endoglin bindingsequence is present. Measurement binding affinity, association rates,disassociation rates and avidity can be accomplished usingart-recognized assays including (Surface Plasmon Resonance), but notlimited to, an enzyme-linked-immunosorbent assay (ELISA), ScatchardAnalysis, BIACORE analysis, etc., as well as other assays commonly usedand known to those of ordinary skill in the art.

Measurement of binding of antibodies to endoglin and/or the ability ofthe antibodies and antigen-binding fragments thereof, for example, toinhibit fibrosis, can be determined using, for example, anenzyme-linked-immunosorbent assay (ELISA), a competitive binding assay,an ELISPOT assay, or any other useful assay known in the art. Theseassays are commonly used and well-known to those of ordinary skill inthe art.

In one non-limiting embodiment, an ELISA assay can be used to measurethe binding capability of specific antibodies or antigen-bindingfragments that bind to endoglin.

Assays, such as an ELISA, also can be used to identify antibodies orantigen-binding fragments thereof which exhibit increased specificity incomparison to other antibodies or antigen-binding fragments thereof.Assays, such as an ELISA, also can be used to identify antibodies orantigen-binding fragments thereof with bind to epitopes across one ormore polypeptides and across one or more species of endoglin. Thespecificity assay can be conducted by running parallel ELISAs in which atest antibodies or antigen-binding fragments thereof is screenedconcurrently in separate assay chambers for the ability to bind one ormore epitopes on different species of the polypeptide containing theendoglin epitopes to identify antibodies or antigen-binding fragmentsthereof that bind to endoglin. Another technique for measuring apparentbinding affinity familiar to those of skill in the art is a surfaceplasmon resonance technique (analyzed on a BIACORE® 2000 system)(Liljeblad, et al., Glyco. J. 2000, 17:323-329). Standard measurementsand traditional binding assays are described by Heeley, R. P., Endocr.Res. 2002, 28:217-229.

Competitive binding assays for assessing disruption of BMP9 binding to aTGF-beta receptor as described in the Examples may also be used toassess their ability to treat, inhibit, or ameliorate fibrosis.

Humanized and deimmunized antibodies described herein can also beassayed for their ability to treat fibrosis. Any suitable assay known toone of skill in the art can be used to monitor such effects. Severalsuch techniques are described herein. In one example, the antibodies andantigen-binding fragments described herein are assayed for their abilityto bind endoglin. In another example, affinity constants for theantibodies and antigen-binding fragments described herein are determinedby surface plasmon resonance (SPR).

II. Compositions

Each of the compounds described herein can be used as a composition whencombined with an acceptable carrier or excipient. Such compositions areuseful for in vitro or in vivo analysis or for administration to asubject in vivo or ex vivo for treating a subject with the disclosedcompounds.

Thus pharmaceutical compositions can include, in addition to activeingredient, a pharmaceutically acceptable excipient, carrier, buffer,stabilizer or other materials well known to those skilled in the art.Such materials should be non-toxic and should not interfere with theefficacy of the active ingredient. The precise nature of the carrier orother material will depend on the route of administration.

Pharmaceutical formulations comprising a protein of interest, e.g., anantibody or antigen-binding fragment, identified by the methodsdescribed herein can be prepared for storage by mixing the proteinhaving the desired degree of purity with optional physiologicallyacceptable carriers, excipients or stabilizers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the formof lyophilized formulations or aqueous solutions. Acceptable carriers,excipients, or stabilizers are those that are non-toxic to recipients atthe dosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN®, PLURONICS® or polyethylene glycol (PEG).

Other reagents that may be included in a composition described hereinare contemplated in International Application No. PCT/US2013/058265,filed Sep. 5, 2013, which is hereby incorporated by reference is itsentirety.

Acceptable carriers are physiologically acceptable to the administeredpatient and retain the therapeutic properties of the compounds with/inwhich it is administered. Acceptable carriers and their formulations areand generally described in, for example, Remington's pharmaceuticalSciences (18th Edition, ed. A. Gennaro, Mack Publishing Co., Easton, Pa.1990). One exemplary carrier is physiological saline. The phrase“pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject compoundsfrom the administration site of one organ, or portion of the body, toanother organ, or portion of the body, or in an in vitro assay system.Each carrier is acceptable in the sense of being compatible with theother ingredients of the formulation and not injurious to a subject towhom it is administered. Nor should an acceptable carrier alter thespecific activity of the subject compounds.

In one aspect, provided herein are pharmaceutically acceptable orphysiologically acceptable compositions including solvents (aqueous ornon-aqueous), solutions, emulsions, dispersion media, coatings, isotonicand absorption promoting or delaying agents, compatible withpharmaceutical administration. Pharmaceutical compositions orpharmaceutical formulations therefore refer to a composition suitablefor pharmaceutical use in a subject. The pharmaceutical compositions andformulations include an amount of a compound described herein and apharmaceutically or physiologically acceptable carrier.

Compositions can be formulated to be compatible with a particular routeof administration (i.e., systemic or local). Thus, compositions includecarriers, diluents, or excipients suitable for administration by variousroutes.

In another embodiment, the compositions can further comprise, if needed,an acceptable additive in order to improve the stability of thecompounds in composition and/or to control the release rate of thecomposition. Acceptable additives do not alter the specific activity ofthe subject compounds. Exemplary acceptable additives include, but arenot limited to, a sugar such as mannitol, sorbitol, glucose, xylitol,trehalose, sorbose, sucrose, galactose, dextran, dextrose, fructose,lactose and mixtures thereof. Acceptable additives can be combined withacceptable carriers and/or excipients such as dextrose. Alternatively,exemplary acceptable additives include, but are not limited to, asurfactant such as polysorbate 20 or polysorbate 80 to increasestability of the peptide and decrease gelling of the solution. Thesurfactant can be added to the composition in an amount of 0.01% to 5%of the solution. Addition of such acceptable additives increases thestability and half-life of the composition in storage.

The pharmaceutical composition can be administered, for example, byinjection, including, but not limited to, subcutaneous, subcutaneous,intravitreal, intradermal, intravenous, intra-arterial, intraperitoneal,or intramuscular injection. Excipients and carriers for use informulation of compositions for each type of injection are contemplatedherein. The following descriptions are by example only and are not meantto limit the scope of the compositions. Compositions for injectioninclude aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CREMOPHOREL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. Fluidity can be maintained, for example, by the use of acoating such as lecithin, by the maintenance of the required particlesize in the case of dispersion and by the use of surfactants.Antibacterial and antifungal agents include, for example, parabens,chlorobutanol, phenol, ascorbic acid and thimerosal. Isotonic agents,for example, sugars, polyalcohols such as manitol, sorbitol, and sodiumchloride may be included in the composition. The resulting solutions canbe packaged for use as is, or lyophilized; the lyophilized preparationcan later be combined with a sterile solution prior to administration.For intravenous, injection, or injection at the site of affliction, theactive ingredient will be in the form of a parenterally acceptableaqueous solution which is pyrogen-free and has suitable pH, isotonicityand stability. Those of relevant skill in the art are well able toprepare suitable solutions using, for example, isotonic vehicles such asSodium Chloride Injection, Ringer's Injection, Lactated Ringer'sInjection. Preservatives, stabilizers, buffers, antioxidants and/orother additives may be included, as needed. Sterile injectable solutionscan be prepared by incorporating an active ingredient in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive ingredient into a sterile vehicle which contains a basicdispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Compositions can be conventionally administered intravitreally,sub-cutaneous, or via intravitreal implant.

Compositions can be conventionally administered intravenously, such asby injection of a unit dose, for example. For injection, an activeingredient can be in the form of a parenterally acceptable aqueoussolution which is substantially pyrogen-free and has suitable pH,isotonicity and stability. One can prepare suitable solutions using, forexample, isotonic vehicles such as Sodium Chloride Injection, Ringer'sInjection, Lactated Ringer's Injection. Preservatives, stabilizers,buffers, antioxidants and/or other additives may be included, asrequired. Additionally, compositions can be administered viaaerosolization.

In one embodiment, the composition is lyophilized, for example, toincrease shelf-life in storage. When the compositions are considered foruse in medicaments or any of the methods provided herein, it iscontemplated that the composition can be substantially free of pyrogenssuch that the composition will not cause an inflammatory reaction or anunsafe allergic reaction when administered to a human patient. Testingcompositions for pyrogens and preparing compositions substantially freeof pyrogens are well understood to one or ordinary skill of the art andcan be accomplished using commercially available kits.

Acceptable carriers can contain a compound that stabilizes, increases ordelays absorption or clearance. Such compounds include, for example,carbohydrates, such as glucose, sucrose, or dextrans; low molecularweight proteins; compositions that reduce the clearance or hydrolysis ofpeptides; or excipients or other stabilizers and/or buffers. Agents thatdelay absorption include, for example, aluminum monostearate andgelatin. Detergents can also be used to stabilize or to increase ordecrease the absorption of the pharmaceutical composition, includingliposomal carriers. To protect from digestion the compound can becomplexed with a composition to render it resistant to acidic andenzymatic hydrolysis, or the compound can be complexed in anappropriately resistant carrier such as a liposome. Means of protectingcompounds from digestion are known in the art (see, e.g., Fix (1996)Pharm Res. 13:1760 1764; Samanen (1996) J. Pharm. Pharmacol. 48:119 135;and U.S. Pat. No. 5,391,377, describing lipid compositions for oraldelivery of therapeutic agents).

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human.

The term “unit dose” when used in reference to a therapeutic compositionrefers to physically discrete units suitable as unitary dosage forhumans, each unit containing a predetermined quantity of active materialcalculated to produce the desired therapeutic effect in association withthe required diluent; i.e., carrier, or vehicle.

The compositions can be administered in a manner compatible with thedosage formulation, and in a therapeutically effective amount. Thequantity to be administered depends on the subject to be treated,capacity of the subject's immune system to utilize the activeingredient, and degree of binding capacity desired. Precise amounts ofactive ingredient required to be administered depend on the judgment ofthe practitioner and are peculiar to each individual. Suitable regimesfor initial administration and booster shots are also variable, but aretypified by an initial administration followed by repeated doses at oneor more hour intervals by a subsequent injection or otheradministration. Alternatively, continuous intravenous infusionsufficient to maintain concentrations in the blood are contemplated.

One embodiment contemplates the use of the compositions described hereinto make a medicament for treating a condition, disease or disorderdescribed herein. Medicaments can be formulated based on the physicalcharacteristics of the patient/subject needing treatment, and can beformulated in single or multiple formulations based on the stage of thecondition, disease or disorder. Medicaments can be packaged in asuitable package with appropriate labels for the distribution tohospitals and clinics wherein the label is for the indication oftreating a subject having a disease described herein. Medicaments can bepackaged as a single or multiple units. Instructions for the dosage andadministration of the compositions can be included with the packages asdescribed below. The invention is further directed to medicaments of ahumanized and deimmunized anti-endoglin antibody or antigen bindingfragment thereof described hereinabove and a pharmaceutically acceptablecarrier.

Provided herein are compositions of humanized and deimmunized antibodiesand antigen-binding fragments thereof that bind endoglin and includethose such as described elsewhere herein. Humanized and deimmunizedantibodies and antigen-binding fragments thereof as described herein canbe used for the treatment, inhibition, or amelioration of fibrosis.

A composition (an antibody or an antigen-binding fragment describedherein) can be administered alone or in combination with a secondcomposition either simultaneously or sequentially dependent upon thecondition to be treated. In one embodiment, a second therapeutictreatment is a fibrotic inhibitor (as described herein). When two ormore compositions are administered, the compositions can be administeredin combination (either sequentially or simultaneously). For example acombination treatment could include perfinedone or a VEGF receptortyrosine kinase inhibitor. It would be understood that otherart-recognized drugs or compounds could be administered with theantibodies described herein in combination therapy. A composition can beadministered in a single dose or multiple doses.

In one embodiment of the present invention, the compositions areformulated to be free of pyrogens such that they are acceptable foradministration to human patients. Testing compositions for pyrogens andpreparing pharmaceutical compositions free of pyrogens are wellunderstood to one of ordinary skill in the art.

One embodiment of the present invention contemplates the use of any ofthe compositions of the present invention to make a medicament fortreating a disorder of the present invention. Medicaments can beformulated based on the physical characteristics of the patient/subjectneeding treatment, and can be formulated in single or multipleformulations based on the disorder. Medicaments of the present inventioncan be packaged in a suitable pharmaceutical package with appropriatelabels for the distribution to hospitals and clinics wherein the labelis for the indication of treating a disorder as described herein in asubject. Medicaments can be packaged as a single or multiple units.Instructions for the dosage and administration of the pharmaceuticalcompositions of the present invention can be included with thepharmaceutical packages.

III. Methods of Use

Provided herein is a method of inducing a response in a patient (humanor non-human) by administering to the patient a composition of ahumanized and deimmunized antibody or antigen-binding fragment thereofthat preferentially binds to endoglin. The binding site to which theantibody binds can be a continuous or conformation/dis-continuousepitope.

An effective response of the present invention is achieved when thepatient experiences partial or total alleviation or reduction of signsor symptoms of illness, and specifically includes, without limitation,prolongation of survival. The expected progression-free survival timesmay be measured in months to years, depending on prognostic factorsincluding the number of relapses, stage of disease, and other factors.Prolonging survival includes without limitation times of at least 1month (mo), about at least 2 mos., about at least 3 mos., about at least4 mos., about at least 6 mos., about at least 1 year, about at least 2years, about at least 3 years, etc. Overall survival can be alsomeasured in months to years. Alternatively, an effective response may bethat a patient's symptoms remain static. Further indications oftreatment of indications are described in more detail below.

Compositions of antibodies and antigen-binding fragments describedherein can be used as non-therapeutic agents (e.g., as affinitypurification agents). Generally, in one such embodiment, a protein ofinterest is immobilized on a solid phase such a Sephadex resin or filterpaper, using conventional methods known in the art. The immobilizedprotein is contacted with a sample containing the target of interest (orfragment thereof) to be purified, and thereafter the support is washedwith a suitable solvent that will remove substantially all the materialin the sample except the target protein, which is bound to theimmobilized antibody. Finally, the support is washed with anothersuitable solvent, such as glycine buffer, pH 5.0, which will release thetarget protein. In addition to purification, compositions can be usedfor treatment of fibrosis.

The term “contacting” as used herein refers to adding together asolution or composition of a compound with a liquid medium bathing thepolypeptides, cells, tissue or organ from an organism. Alternately,“contacting” refers to mixing together a solution or composition of acompound, with a liquid such as blood, serum, or plasma derived from anorganism. For in vitro applications, a composition can also compriseanother component, such as dimethyl sulfoxide (DMSO). DMSO facilitatesthe uptake of the compounds or solubility of the compounds. The solutioncomprising the test compound may be added to the medium bathing thecells, tissues, or organs, or mixed with another liquid such as blood,by utilizing a delivery apparatus, such as a pipette-based device orsyringe-based device. For in vivo applications, contacting can occur,for example, via administration of a composition to a patient by anysuitable means; compositions with pharmaceutically acceptable excipientsand carriers have been described in more detail above.

A “subject” (e.g., a mammal such as a human or a non-human animal suchas a primate, rodent, cow, horse, llama, alpaca, pig, sheep, etc.)according to one embodiment of the present application, is a mammal whoexhibits one or more clinical manifestations and/or symptoms offibrosis. In certain situations, the patient may be asymptomatic and yetstill have clinical manifestations of fibrosis. An antibody orantigen-binding fragment thereof can be conjugated to a therapeuticmoiety or be a fusion protein containing a therapeutic moiety. Affinitytags such as, for example, His6 tags (SEQ ID NO: 85) are conventional inthe art.

Antibodies or antigen-binding fragments thereof provided herein are suchthat they can be conjugated or linked to a therapeutic moiety and/or anaffinity tag. Methods for conjugating or linking polypeptides are wellknown in the art. Associations (binding) between compounds and labelsinclude any means known in the art including, but not limited to,covalent and non-covalent interactions, chemical conjugation as well asrecombinant techniques.

A. Binding of Endoglin and Fibrosis

Endoglin (CD105) is expressed on the cell surface as a 180 kDahomodimeric transmembrane protein. The external domain binds TGF-β1 and-3 isoforms with high affinity (50 nM), and the transmembrane and theintracellular domains of CD105 share a 71% sequence similarity with betaglycan. The human CD105 gene is located on chromosome 9q34, identifiedusing fluorescence in situ hybridization, and the coding region contains14 exons, and two different isoforms (L and S) of CD105 with capacity tobind TGF-β have been characterized. The L-CD105 consists of 633 aminoacid residues with 47 amino acid residues in the cytoplasmic tail asopposed to the S-CD105, which consists of 600 amino acid residues with a14 amino acid cytoplasmic tail. However, L-CD105 is the predominantform. CD105 is constitutively phosphorylated in endothelial cells,mainly on serine and threonine residues, and this phosphorylation is dueto the constitutively active TGF-β RII within the cell. The human CD105amino acid sequence contains the tripeptide arginine-glycine-asparticacid (RGD) located in an exposed region of the extracellular domain. TheRGD peptide is a key recognition structure found on ECM proteins such asfibronectin, vitronectin, von Willebrand factor (vWF), type I collagen,and fibrinogen and is recognized by cell surface integrins.

CD105 is a member of the TGF-β receptor family that is expressed byproliferating endothelial cells. Normal levels of CD105 are needed forendothelial cell proliferation. CD105 expression is increased bycellular hypoxia through the production of hypoxia-inducible factor-1-α(HIF-1-α) and protects hypoxic cells from apoptosis. Several functionsof CD105 are associated with TGF-β signaling. TGF-β signals throughheterodimeric receptors consisting of serine kinases, receptor I (RI),and receptor II (RII). Binding of TGF-β to the external domains of thereceptor unmasks the cytoplasmic RII kinase activity that phosphorylatesthe TGF-β RI, which can then interact with downstream signalers such asthe Smad proteins. CD105 forms part of the TGF-β receptor complex but itcan exist independently on the cell surface. In endothelial cells,binding of TGF-β to endoglin and the RI and RII receptors inhibits cellproliferation through the phosphorylation of the SMAD 2 and 3intracellular proteins (Nolan-Stevaux, 2012) Thus, CD105 modulates TGF-βfunctions via interaction with TGF-β RI and TGF-β RII and modifies thephosphorylation of downstream Smad proteins.

CD105 also binds other growth factors such as activin A and bonemorphogenic proteins (BMP)-10, -9, -7 and -2. Binding of BMP-9 to acomplex consisting of endoglin, the BMP receptor II and ALK1 is criticalto the process of SMAD 1/5/8 phosphorylation that is required for theproliferation of endothelial cells into blood vessels (i.e., the processof angiogenesis [Nolan-Stevaux, 2012]. Antibodies that bind to endoglinto inhibit angiogenesis inhibit BMP binding. TRC105 binds to theendoglin orphan domain to competitively inhibit BMP binding and inhibitSMAD 1/5/8 phosphorylation and inhibit angiogenesis. Antibodies that donot inhibit BMP binding do not inhibit angiogenesis in primaryendothelial cells.

The sequences of human and mouse CD105 are not identical. TRC105 bindsto human endoglin and competitively inhibits human BMP binding. TRC105also binds to mouse endoglin. However, TRC105 dose not prevent thebinding of mouse BMP to mouse endoglin. The antibody M1043 binds tomouse endoglin to competitively inhibit mouse BMP binding. M1043therefore can be used in mouse models to mimic the expected effects ofTRC105 in humans.

In addition to their use for purification of endoglin, these humanizedand deimmunized antibodies are useful for detection and diagnosticpurposes as well as therapeutic purposes. The antibodies provided hereincan be used for the formulation of medicaments for the treatment avariety of fibrotic conditions and diseases.

Murine monoclonal antibodies (mAbs) have been raised against endoglinwhich modulate endoglin activity an. These murine antibodies aredescribed in U.S. Pat. Nos. 5,928,641, 6,200,566, 6,190,660, and7,097,836, each of which is hereby incorporated in their entirety.Additionally, the ex vivo and in vivo efficiency of a number of theseantibodies has been demonstrated; monoclonal antibodies that bindendoglin are of interest as endoglin modulating compounds. Therapeuticuse of murine antibodies is not feasible, however, as administration ofthe murine antibodies have a number of limitations, includingimmunogenicity in, for example, the form of human anti-mouse antibodies(HAMA).

“Fibrosis” is used herein to refer to abnormal accumulation of fibroustissue that can occur as a part of the wound-healing process in damagedtissue. Such tissue damage may result from physical injury,inflammation, infection, exposure to toxins, and other causes. Examplesof fibrosis include, but are not limited to, dermal scar formation,keloids, liver fibrosis, pulmonary fibrosis, kidney fibrosis, cardiacfibrosis, scleroderma, and glomerulosclerosis. Other fibrotic conditionsand disease are discussed in more detail below.

The term “fibrosis inhibitory composition” refers to a composition whichinhibits a fibrosis-mediated process.

Fibroblast differentiation is associated with a variety of fibroticdiseases including but not limited to scleroderma, keloid scarring,rheumatoid arthritis, lupus, nephrogenic fibrosing dermopathy, andidiopathic pulmonary fibrosis. They play a role in the formation offibrotic lesions after Schistosoma japonicum infection in mice and arealso implicated in fibrosis associated with autoimmune diseases.Fibroblasts have also been implicated in pathogenic fibrosis associatedwith radiation damage, Lyme disease and pulmonary fibrosis, as well asstromal remodeling in pancreatitis and stromal fibrosis, whereas lack ofsuch fibroblasts is associated with pancreatic tumors andadenocarcinomas. Fibrosis additionally occurs in asthma patients andpossibly other pulmonary diseases such as chronic obstructive pulmonarydisease when fibrocytes undergo further differentiation intomyofibroblasts. Fibrosis involves the unchecked proliferation anddifferentiation of fibroblasts.

C. Treatment, Inhibition or Amelioration of Fibrosis with Humanized andDeimmunized Anti-Endoglin Antibodies

CD105 acts to modulate signaling of multiple kinase receptor complexesof the TGF-β superfamily, including TGF-β receptors (TGF-βR), BMPreceptors, activin receptor-like kinases (ALK) and activin receptors. Inendothelial cells, TRC105 blocks BMP binding to human CD105 and causesthe SMAD 1/5/8 pathway to be blocked or down-regulated. Decreasedphosphorylated SMAD 1/5/8 allows unopposed phospho-SMAD 2/3 signaling torestore a quiescent phenotype in human endothelial cells.

These observations led the present inventor to consider that antibodiesto endoglin that inhibit the BMP signaling pathway could interrupt SMAD1/5/8 signaling in fibroblasts to inhibit fibrosis. The importance ofBMP signaling in promoting the process of fibrosis could be demonstratedin mouse models using the IgG1 antibody M1043. The treatment of humanfibrosis could use the humanized and deimmunized antibody TRC205 (SEQ IDNO 89/SEQ ID NO 93; IgG4) to interrupt SMAD 1/5/8 signaling in humanfibroblasts to inhibit fibrosis.

TRC205 has an advantage of being an IgG4 antibody and, therefore, willnot engage natural killer (NK) and other effector cells that could causeinflammation that might promote fibrosis.

TRC105 and TRC205 would be expected to be active in mouse models throughtheir ability to bind mouse endoglin and cause solubilization of theendoglin receptor. Soluble endoglin in plasma binds to BMP9 and makesBMP unavailable to bind to endoglin on the cell surface of fibroblastsand other cells, to initiate cell signaling. All three endoglinantibodies, M1043, TRC105 and TRC105, may also inhibit TGF-betasignaling through TGF-beta binding by soluble endoglin shed as a resultof endoglin antibody binding to TGF-beta.

Provided herein are methods for treating, inhibiting, or amelioratingfibrosis comprising administering a composition comprising a humanizedand deimmunized antibody or antigen-binding fragment described hereinthat binds to endoglin associated with the disease or disorder andprevents fibrosis (i.e., preventing, treating, ameliorating, orlessening the severity of fibrosis).

Provided herein are methods for treating, inhibiting, or amelioratingtreating fibrosis, comprising administering a composition comprising ahumanized and deimmunized antibody or antigen-binding fragment describedherein that binds to endoglin treats fibrosis.

Provided herein are methods for treating, inhibiting, or amelioratingtreating fibrosis, comprising administering a composition comprising ahumanized and deimmunized antibody or antigen-binding fragment describedherein that binds to a endoglin receptor and blocks BMP9 binding toendoglin.

Provided herein are methods for treating, inhibiting, or amelioratingtreating fibrosis, comprising administering a composition comprising ahumanized and deimmunized antibody or antigen-binding fragment describedherein that binds to endoglin receptor and inhibits Smad 1/5/8signaling.

As used herein, “amelioration” “inhibition,” “treatment” and “treating”refer to, for example, stasis of symptoms, prolongation of survival,partial or full amelioration of symptoms, and partial or fulleradication of a condition, disease or disorder associated withfibrosis.

In one embodiment, fibrosis is inhibited by about 5%, about 10%, about15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%,about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about80%, about 85%, about 90%, about 95%, or about 100%, following treatmentwith one or more doses of a humanized and deimmunized antibody comparedto treatment with a placebo or compared to a subject that does notreceive any treatment.

In another embodiment, symptoms of fibrosis may be ameliorated by about5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%,about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about100%, following treatment with one or more doses of a humanized anddeimmunized antibody compared to treatment with a placebo or compared toa subject that does not receive any treatment.

Treatment also refers to resolution of one or more symptoms of fibrosis.Treatment also refers to stasis of symptoms where fibrosis in a subjectdoes not progress.

Pain associated with fibrosis may be reduced by about 5%, about 10%,about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%,about 80%, about 85%, about 90%, about 95%, or about 100%, followingtreatment with one or more doses of a humanized and deimmunized antibodycompared to treatment with a placebo or compared to a subject that doesnot receive any treatment.

Other means of assessing patients for various fibrotic conditions anddiseases are discussed below. In some instances, the tests described maybe administered to determine the extent of treatment; a doctor maydetermine based upon the results of the tests whether to continue ordiscontinue treatment, increase or decrease dosage of the antibodies, orany combination thereof.

Compositions can be administered to a patient in a therapeuticallyeffective amount, i.e., that are effective for producing some desiredtherapeutic effect by inhibiting fibrosis such as described herein whichcan be associated with endoglin, at a reasonable benefit/risk ratioapplicable to any medical treatment. For the administration of thepresent compositions to human subjects, the compositions can beformulated by methodology known by one of ordinary skill in the art. Atherapeutically effective amount is an amount achieves at leastpartially a desired therapeutic or prophylactic effect in an organ ortissue. The amount of a humanized and deimmunized anti-endoglin antibodyor antigen binding fragment thereof necessary to bring about preventionand/or therapeutic treatment of fibrosis is not fixed per se. The amountof humanized and deimmunized anti-endoglin antibody or antigen bindingfragment thereof administered may vary with the type of fibrosis,extensiveness of the fibrosis, and size of the mammal suffering fromfibrosis. In one embodiment, two or more humanized anti-endoglinantibodies described herein are administered to a patient incombination. Combination includes concomitant or subsequentadministration of the antibodies.

“Administering” is defined herein as a means providing the compositionto the patient in a manner that results in the composition being insidethe patient's body. Such an administration can be by any routeincluding, without limitation, locally, regionally or systemically bysubcutaneous, intravitreal, intradermal, intravenous, intra-arterial,intraperitoneal, or intramuscular administration (e.g., injection).“Concurrent administration” means administration within a relativelyshort time period from each other; such time period can be less than 2weeks, less than 7 days, less than 1 day and could even be administeredsimultaneously.

Actual dosage levels of the active ingredients in the compositions canbe varied so as to obtain an amount of the active ingredient that iseffective to achieve the desired therapeutic response for a particularpatient, composition, and mode of administration, without being toxic tothe patient. The selected dosage level will depend upon a variety offactors including the activity of the particular compound employed, theroute of administration, the time of administration, the rate ofexcretion of the particular compound being employed, the duration of thetreatment, other drugs, compounds and/or materials used in combinationwith the particular composition employed, the age, sex, weight,condition, general health and prior medical history of the patient beingtreated, and like factors well known in the medical arts. The antibodiesand antigen-binding fragments described herein can be administered to asubject in various dosing amounts and over various time frames.Non-limiting doses include about 0.01 mg/kg, about 0.05 mg/kg, about 0.1mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg,about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 60mg/kg, about 70 mg/kg, about 80 mg/kg, about 90 mg/kg, about 100 mg/kg,about 125 mg/kg, about 150 mg/kg, about 175 mg/kg, about 200 mg/kg perpatient, or any integer in between. Additionally, the dose(s) of anantibody or antigen-binding fragment can be administered twice a week,weekly, every two weeks, every three weeks, every 4 weeks, every 6weeks, every 8 weeks, every 12 weeks, or any combination of weekstherein. Dosing cycles are also contemplated such as, for example,administering antibodies or antigen-binding fragments thereof once ortwice a week for 4 weeks, followed by two weeks without therapy.Additional dosing cycles including, for example, different combinationsof the doses and weekly cycles described herein are also contemplatedwithin the invention.

“Contacting” is defined herein as a means of bringing a composition asprovided herein in physical proximity with a cell, organ, tissue orfluid as described herein. Contacting encompasses systemic or localadministration of any of the compositions provided herein and includes,without limitation, in vitro, in vivo and/or ex vivo procedures andmethods. “Combining” and “contacting” are used interchangeably hereinand are meant to be defined in the same way.

A response is achieved when the patient experiences partial or totalalleviation, or reduction of signs or symptoms of illness, andspecifically includes, without limitation, prolongation of survival. Theexpected progression-free survival times can be measured in months toyears, depending on prognostic factors including the number of relapses,stage of disease, and other factors. Prolonging survival includeswithout limitation times of at least 1 month (mo), about at least 2months (mos.), about at least 3 mos., about at least 4 mos., about atleast 6 mos., about at least 1 year, about at least 2 years, about atleast 3 years, or more. Overall survival can also be measured in monthsto years. The patient's symptoms can remain static or can decrease.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount (ED50) of the compositionrequired. For example, the physician or veterinarian could start dosesof the compounds employed in the composition at levels lower than thatrequired in order to achieve the desired therapeutic effect andgradually increase the dosage until the desired effect is achieved.Alternatively, a dose can remain constant.

Compositions can be administered to a patient by any convenient routesuch as described above. Regardless of the route of administrationselected, the compounds of the present invention, which can be used in asuitable hydrated form, and/or the compositions, are formulated intoacceptable dosage forms such as described below or by other conventionalmethods known to those of skill in the art.

Antibodies can be combined with a therapeutic moiety using methods knownin the art such as, for example, chemical conjugation, covalent ornon-covalent bonds or recombinant techniques to create conjugates orfusion proteins such as described in more detail below. Alternatively,antibodies and/or other agents can be combined in separate compositionsfor simultaneous or sequential administration.

Toxicity and therapeutic efficacy of such ingredient can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.While compounds that exhibit toxic side effects may be used, care shouldbe taken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage to healthycells and, thereby, reduce side effects.

Data obtained from cell culture assays and animal studies can be used informulating a range of dosage for use in humans. The dosage of suchcompounds lies preferably within a range of circulating concentrationsthat include the ED₅₀ with little or no toxicity. The dosage may varywithin this range depending upon the dosage form employed and the routeof administration utilized. For any compound used in the method of theinvention, the therapeutically effective dose can be estimated initiallyfrom cell culture assays. A dose can be formulated in animal models toachieve a circulating plasma concentration arrange that includes theIC₅₀ (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition) as determined in cell culture. Levels in plasmacan be measured, for example, by high performance liquid chromatography.Such information can be used to more accurately determine useful dosesin humans.

The unique specificity of the antibodies which recognize (e.g., bind) anepitope on endoglin and provide therapeutic uses for fibrosis such asdescribed herein.

In another aspect, methods are provided for inhibiting, treating, orameliorating fibrosis in a subject in vivo, comprising administering tothe subject an effective amount of an anti-endoglin antibody describedherein.

Liver (Hepatic) Fibrosis

Fibrosis of the liver is implicated in the pathology of numerous hepaticdiseases. As previously noted, fibrosis occurs as a complication ofhaemochromatosis, Wilson's disease, alcoholism, schistosomiasis, viralhepatitis, bile duct obstruction, exposure to toxins, and metabolicdisorders. Left unchecked, hepatic fibrosis progresses to cirrhosis(defined by the presence of encapsulated nodules), liver failure, anddeath.

Liver (hepatic) fibrosis, for example, occurs as a part of thewound-healing response to chronic liver injury. Fibrosis occurs as acomplication of haemochromatosis, Wilson's disease, alcoholism,schistosomiasis, viral hepatitis, bile duct obstruction, exposure totoxins, and metabolic disorders. This formation of scar tissue isbelieved to represent an attempt by the body to encapsulate the injuredtissue. Liver fibrosis is characterized by the accumulation ofextracellular matrix that can be distinguished qualitatively from thatin normal liver. Left unchecked, hepatic fibrosis progresses tocirrhosis (defined by the presence of encapsulated nodules), liverfailure, and death.

Liver fibrosis includes, but is not limited to, cirrhosis (e.g., primarybiliary cirrhosis (PBC), biliary cirrhosis), and associated conditionssuch as chronic viral hepatitis, hepatitis C viral (HCV) infection,hepatitis B viral (HBV) infection, Alcoholic liver disease (ALD),Primary sclerosing cholangitis, Hereditary hemochromatosis, and Wilson'sdisease, non-alcoholic fatty liver disease (NAFLD), alcoholicsteatohepatitis (ASH), non-alcoholic steatohepatitis (NASH), a HumanImmunodeficiency Virus (HIV), schistosomiasis and autoimmune hepatitis.

Chronic insults to the liver from such sources as parasites and viralinfection (e.g. HBV, HCV, HIV, schistosomiasis) or the long term stressfrom alcohol consumption typically result in remodeling of the liver,presumably to encapsulate the damaged area and protect the remainingliver tissue from damage. Liver fibrosis results in extracellular matrixchanges, including 3-10 fold increases in total collagen content andreplacement of the low density basement membrane with high-densitymatrix, which impair the metabolic and synthesis function ofhepatocytes, hepatic stellate cells and endothelial cells.

Nonalcoholic steatohepatitis (NASH) is a common, often “silent” liverdisease. It resembles alcoholic liver disease, but occurs in people whodrink little or no alcohol. The major feature in NASH is fat in theliver, along with inflammation and damage. Most people with NASH feelwell and are not aware that they have a liver problem. Nevertheless,NASH can be severe and can lead to cirrhosis, in which the liver ispermanently damaged and scarred and no longer able to work properly.

The efficacy of administration of a composition described herein fortreatment, inhibition or amelioration of NASH can be assessed using artrecognized tests including, for example, those described in Example 11.

The efficacy of administration of a composition described herein fortreatment, inhibition or amelioration of cirrhosis can be assessed usingart recognized tests including, for example, ultrasound, elastographytechniques, abdominal CT scan, liver/bile duct MRI (MRCP), serologytests for hepatitis viruses, testing for autoantibodies, and endoscopy(gastroscopy). The severity of cirrhosis can be classified with aChild-Pugh score which uses bilirubin, albumin, INR, presence andseverity of ascites, and encephalopathy to classify patients in class A,B, or C. Class A has a favorable prognosis, while class C is at highrisk of death. The Model for End-Stage Liver Disease (MELD) score andthe Pediatric End-Stage Liver Disease (PELD) score may also be used tograde the severity of cirrhosis.

The compositions described herein are thus useful for treating,inhibiting, or ameliorating fibrotic liver diseases such as thosedescribed herein.

Kidney Fibrosis

Like liver fibrosis, kidney fibrosis can result from various diseasesand insults to the kidneys. Examples of such diseases and insultsinclude chronic kidney disease, metabolic syndrome, vesicoureteralreflux, tubulointerstitial renal fibrosis, diabetes (including diabeticnephropathy), and resultant glomerular nephritis (GN), including, butnot limited to, focal segmental glomerulosclerosis and membranousglomerulonephritis, mesangiocapillary GN.

It has become recognized that metabolic syndrome is a cluster ofabnormalities including diabetic hallmarks such as insulin resistance,as well as central or visceral obesity and hypertension. In nearly allcases, dysregulation of glucose results in the stimulation of cytokinerelease and upregulation of extracellular matrix deposition. Additionalfactors contributing to chronic kidney disease, diabetes, metabolicsyndrome, and glomerular nephritis include hyperlipidemia, hypertension,and proteinuria, all of which result in further damage to the kidneysand further stimulate the extracellular matrix deposition. Thus,regardless of the primary cause, insults to the kidneys may result inkidney fibrosis and the concomitant loss of kidney function. Thecompositions described herein are thus useful for the prevention,treatment, and/or amelioration of fibrotic kidney diseases (chronickidney disease, diabetic nephropathy, glomerular nephritis, metabolicsyndrome), and such use is contemplated herein.

Kidney fibrosis includes, but is not limited to, diabetic nephropathy,vesicoureteral reflux, tubulointerstitial renal fibrosis,glomerulonephritis or glomerular nephritis (GN), focal segmentalglomerulosclerosis, membranous glomerulonephritis, or mesangiocapillaryGN.

The efficacy of administration of a composition described herein fortreatment, inhibition or amelioration of including diabetic nephropathycan be assessed using art recognized tests including, for example, themeasurement of urinary albumin. Normoalbuminuria refers to urinaryalbumin excretion <30 mg/24 h, it is the physiological state;Microalbuminuria refers to urinary albumin excretion in the range of30-299 mg/24 h; and Clinical (overt) albuminuria refers to urinaryalbumin excretion ≥300 mg/24 h.

The efficacy of administration of a composition described herein fortreatment, inhibition or amelioration of resultant glomerular nephritis(GN) can be assessed using art recognized tests including, for example,monitoring edema in a subject that has increased protein in the urineand decreased protein in the blood, with increased fat in the blood.Concentrations of proteins in urine, oncotic pressure of the blood andhyperlipidemia can also be monitored to assess treatment.

The efficacy of administration of a composition described herein fortreatment, inhibition or amelioration of vesicoureteral reflux can beassessed using art recognized tests including, for example, Nuclearcystogram (RNC); Fluoroscopic voiding cystourethrogram (VCUG);Ultrasonic cystography; and ultrasound.

The efficacy of administration of a composition described herein fortreatment, inhibition or amelioration of tubulointerstitial renalfibrosis can be assessed using art recognized tests including, forexample, tests to assess hyperkalemia, metabolic acidosis, and kidneyfailure. Blood may be collected and tested for eosinophilia as about 23%of patients have eosinophilia. Urinary samples may also be assessed foreosinophiluria; isosthenuria; hematuria; sterile pyuria; andnephrotic-range proteinuria.

The compositions described herein are thus useful for treating,inhibiting, or ameliorating fibrotic kidney diseases such as thosedescribed herein.

Pulmonary Fibrosis

Pulmonary fibrosis refers to, for example, a respiratory disease inwhich scars are formed in the lung tissues, leading to serious breathingproblems. Scar formation and the accumulation of excess fibrousconnective tissue leads to thickening of the walls and causes reducedoxygen supply in the blood. Exemplary diseases include, but are notlimited to, idiopathic pulmonary fibrosis (IPF), idiopathic interstitialpneumonia, and acute respiratory distress syndrome (ARDS). Lung fibrosismay also include, but not be limited to, cryptogenic fibrosingalveolitis, chronic fibrosing interstitial pneumonia, interstitial lungdisease (ILD), chronic obstructive pulmonary disease (COPD), chronicasthma, and diffuse parenchymal lung disease (DPLD).

Chronic pulmonary fibrosis of known and idiopathic origin presentsextraordinary clinical challenges for which treatment options showlimited effectiveness or toxicity, and the median survival ratefollowing diagnosis has changed little. Known profibrotic stimuliinclude radiation, inhaled mineral and organic particles, gaseousoxidants, pharmaceutics and infectious organisms, whereas debatepersists regarding the identity of etiological factors that initiate theclinicopathologic entities of idiopathic interstitial pneumonias (TIP).TIP are a diverse group of disorders involving the distal pulmonaryparenchyma, which share numerous features, but are felt to besufficiently different to justify designation as separate disorders.Their pathogenesis remains unclear but is thought to center around aninjury (or multiple injuries) to the lung followed by attempts to healthis injury. Fibroblastic foci, small aggregates of activelyproliferating fibroblasts, are believed to represent the organization ofprior foci of injury and indicate that fibrosis is active and ongoing.

Chronic pulmonary fibrosis results from scarring throughout the lungswhich can be caused by many conditions including chronic inflammatoryprocesses (sarcoidosis, Wegener's granulomatosis), infections,environmental agents (asbestos, silica, exposure to certain gases),exposure to ionizing radiation (such as radiation therapy to treattumors of the chest), chronic conditions (lupus, rheumatoid arthritis),and even certain medications. In a condition known as hypersensitivitypneumonitis, fibrosis of the lung can develop following a heightenedimmune reaction to inhaled organic dusts or occupational chemicals. Thiscondition most often results from inhaling dust contaminated withbacterial, fungal, or animal products.

The compositions described herein are thus useful for treating,inhibiting, or ameliorating fibrotic pulmonary diseases such as thosedescribed herein.

The efficacy of administration of a composition described herein can beassessed using art recognized tests including, for example, Spirometry.The modified British Medical Research Council questionnaire (mMRC) orCOPD assessment test (CAT) are questionnaires that may be used todetermine the severity of symptoms. Scores on CAT range from 0-40 withthe higher the score, the more severe the disease. Spirometry may helpto determine the severity of airflow limitation. This is typically basedon the FEV1 or forced vital capacity (FVC) expressed as a percentage ofthe predicted “normal” for the person's age, gender, height and weight.Both the American and European guidelines recommended partly basingtreatment recommendations on the FEV1. The GOLD guidelines suggestdividing people into four categories based on symptoms assessment andairflow limitation. Weight loss and muscle weakness, as well as thepresence of other diseases, may also be assessed.

Recognition and monitoring of symptoms of idiopathic pulmonary fibrosis(IPF) can be used to determine efficacy of the compositions describedherein. A chest x-ray may be utilized at various stages of treatment.

For subjects having acute respiratory distress syndrome (ARDS), anarterial blood gas analysis and chest X-ray may be used for monitoring.

Cardiac Fibrosis

Cardiac fibrosis includes, but is not limited to, congestive heartfailure, heart failure with preserved ejection fraction, cardiomyopathy,post-myocardial infarction defects in heart function; atherosclerosis;rheumatoid arthritis; glaucoma; age-related macular degeneration (wetAMD and dry AMD); emphysema, multiple sclerosis; and chronic asthma mayalso be prevented, treated, or ameliorated with compositions ofdescribed herein. The pathological cardiac condition or disease may behypertension, hypertensive heart disease (HHD), myocardial infarction(MI), acute myocardial infarction, atherosclerosis, or restenosis.

The efficacy of administration of a composition described herein fortreatment, inhibition or amelioration of cardiac fibrosis may beassessed using one or more of the following: measuring cardiac output inthe subject; measuring stroke volume in the subject; measuring meansystolic ejection rate in the subject; measuring systolic blood pressurein the subject; measuring left ventricular ejection fraction in thesubject; determining stroke index in the subject; determining cardiacindex in the subject; measuring left ventricular percent fractionalshortening in the subject; measuring mean velocity of circumferentialfiber shortening in the subject; measuring left ventricular inflowvelocity pattern in the subject; measuring pulmonary venous flowvelocity pattern in the subject; and/or measuring peak early diastolicvelocity of the mitral annulus of the subject.

Exemplary experimental animal models for the testing the compositionsdescribed herein include, but are not limited to, a pressureoverload-induced hypertrophy, an isoproterenol-induced cardiachypertrophy model, an exercise-induced cardiac hypertrophy model, ahigh-salt diet-induced cardiac hypertrophy model, and a hormone-inducedcardiac hypertrophy model.

The compositions described herein are thus useful for treating,inhibiting, or ameliorating cardiac kidney diseases such as thosedescribed herein.

Dermal Scar and Keloid Formation

Dermal scar and keloid formation are known to involve excessive collagendeposition and/or dysregulation of collagen deposition. This deviationfrom normal fibroblast remodeling of injured dermal tissue can result inthick and unsightly scarring. Keloids are known to be, in part, theresult of dysregulated wound healing and subsequent elevated collagendeposition. Keloids, unlike the scars seen in normal wound healing, donot fade or regress over time. Though keloids are typically benigndermal tumors, they are unsightly and can accumulate into moreproblematic skin deformations and/or lesions. The accumulation ofcollagen in the skin is also implicated in scleroderma, a generalizedterm for numerous conditions of thickening or hardening of dermaltissue, where the common element is the overproduction or dysregulationof collagen in the dermal tissues by fibroblasts.

The compositions described herein are thus useful for treating,inhibiting, or ameliorating dermal scar and keloid formation.

Neurofibromatosis

Neurofibromatosis is a genetic disease that causes fibrotic tumors toform within nerve tissue anywhere in the body. Neurofibromatosisincludes neurofibromatosis type 1, which is most common,neurofibromatosis type 2, and Schwannomatosis. The disease can bedisfiguring due to the occurrence of multiple cutaneous and subcutaneousfibrotic lesions throughout the body. Benign tumors may affect hearing,sight, balance, strength and sensation. Neurofibromas may alsodegenerate into malignant tumors that are difficult to treat.

Provided herein are methods of treating, inhibiting, or amelioratingneurofibromatosis with a composition described herein.

Combination Therapy

One would understand that the anti-endoglin antibodies can be effectivefor treating fibrosis, it is contemplated herein that a subject can alsobe treated with one or more additional fibrotic inhibitors.

The term “fibrotic inhibitor” or “anti-fibrotic agent” is used herein,for purposes of the specification and claims, to mean a compound ormolecule including, but not limited to, commercially availableinhibitors. In one embodiment, the one or more additional anti-fibroticagents or anti-fibrotic treatments include, but are not limited to,removal of the underlying cause (e.g., toxin or infectious agent),suppression of inflammation (using, e.g., corticosteroids such asPrednisone, IL-1 receptor antagonists, or other agents), down-regulationof stellate cell activation using, e.g., gamma interferon orantioxidants), promotion of matrix degradation, or promotion of stellatecell apoptosis.

IV. Packages and Kits

In still further embodiments, the present application concerns kits foruse with the compounds described above. Humanized and deimmunizedantibodies, or antigen-binding fragments, that bind endoglin can beprovided in a kit. The kit can, optionally, include one or moreanti-fibrotic reagents. The kits will thus comprise, in suitablecontainer means, a composition comprising an antibody or antigen-bindingfragment thereof that binds endoglin. The kit may comprise an antibodyor antigen-binding fragment thereof that binds endoglin in suitablecontainer means.

The container means of the kits will generally include at least onevial, test tube, flask, bottle, syringe and/or other container means,into which the at least one reagent can be placed, and/or preferably,suitably aliquoted. The kits can include a means for containing anyreagent containers in close confinement for commercial sale. Suchcontainers may include injection and/or blow-molded plastic containersinto which the desired vials are retained. Kits can also include printedmaterial for use of the materials in the kit.

Packages and kits can additionally include a buffering agent, apreservative and/or a stabilizing agent in a pharmaceutical formulation.Each component of the kit can be enclosed within an individual containerand all of the various containers can be within a single package.Invention kits can be designed for cold storage or room temperaturestorage.

Additionally, the preparations can contain stabilizers to increase theshelf-life of the kits and include, for example, bovine serum albumin(BSA). Where the compositions are lyophilized, the kit can containfurther preparations of solutions to reconstitute the lyophilizedpreparations. Acceptable reconstitution solutions are well known in theart and include, for example, pharmaceutically acceptable phosphatebuffered saline (PBS).

Packages and kits can further include one or more components for anassay, such as, for example, an ELISA assay. Samples to be tested inthis application include, for example, blood, plasma, and fresh orfrozen tissue sections (e.g., lung, liver, kidney, cardiac, etc.),tissue secretions, urine, lymph, and products thereof. Packages and kitscan further include one or more components for collection of a sample(e.g., a syringe, a cup, a swab, etc.).

Packages and kits can further include a label specifying, for example, aproduct description, mode of administration and/or indication oftreatment. Packages provided herein can include any of the compositionsas described herein. The package can further include a label fortreating fibrosis.

The term “packaging material” refers to a physical structure housing thecomponents of the kit. The packaging material can maintain thecomponents sterilely and can be made of material commonly used for suchpurposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules,etc.). The label or packaging insert can include appropriate writteninstructions. Kits, therefore, can additionally include labels orinstructions for using the kit components in any method of theinvention. A kit can include a compound in a pack, or dispenser togetherwith instructions for administering the compound in a method describedherein.

Instructions can include instructions for practicing any of the methodsdescribed herein including treatment methods. Instructions canadditionally include indications of a satisfactory clinical endpoint orany adverse symptoms that may occur, or additional information requiredby regulatory agencies such as the Food and Drug Administration for useon a human subject.

The instructions may be on “printed matter,” e.g., on paper or cardboardwithin or affixed to the kit, or on a label affixed to the kit orpackaging material, or attached to a vial or tube containing a componentof the kit. Instructions may additionally be included on a computerreadable medium, such as, for example, CD-ROMs, DVDs, flash memorydevices, solid state memory, magnetic disk drives, magnetic tape drives,optical disk drives, cloud computing systems and services, and the like.In some cases, the program and instructions are permanently,substantially permanently, semi-permanently, or non-transitorily encodedon the media.

The embodiments of the compounds and methods of the present applicationare intended to be illustrative and not limiting. Modifications andvariations can be made by persons skilled in the art in light of theabove teachings specifically those that may pertain to alterations inthe antibodies or antigen-binding fragments which bind endoglinsurrounding the described modifications while maintaining near nativefunctionally with respect to binding of endoglin. Therefore, it shouldbe understood that changes may be made in the particular embodimentsdisclosed which are within the scope of what is described.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

EXAMPLES

The application may be better understood by reference to the followingnon-limiting examples, which are provided as exemplary embodiments ofthe application. The following examples are presented in order to morefully illustrate embodiments of the invention and should in no way beconstrued, however, as limiting the broad scope of the application.

Example 1 Generation and Binding of Anti-CD105 Humanized/DeimmunizedAntibodies

Construction, Expression and Purification of Antibodies

All humanized/deimmunized VH and VK region genes were synthesized usinga series of overlapping oligonucleotides that were annealed, ligated andPCR amplified to give full length synthetic V regions. The assembledvariants were then cloned directly into Antitope Ltd.'s pANT expressionvector system for IgG1 heavy chains and kappa light chains.

All combinations of humanized/deimmunized heavy and light chains (i.e.,a total of 24 pairings) were stably transfected into NS0 cells viaelectroporation and selected using 200 nM methotrexate (Sigma Cat. No.M8407). Methotrexate-resistant colonies for each construct were testedfor IgG expression levels using an IgG1 ELISA. The best expressing lineswere selected and frozen under liquid nitrogen. Successful transfectionand clone selection was achieved for all variants and expression levelsof humanized and humanized/deimmunized antibody variants in saturatedstatic cultures are shown in Table 1.

Twenty-four IgG1 variants were therefore purified from NS0 cell culturesupernatants on a Protein A sepharose column (GE Healthcare Cat. No.110034-93) and quantified by OD_(280 nm) using an extinctioncoefficient, Ec_((0.1%))=1.62, based on the predicted amino acidsequence. Approximately 500 μg of each antibody variant was purified andlead variants were analyzed by reducing SDS-PAGE. Briefly, Coomassieblue stained reducing SDS-PAGE gel of lead antibody variants. 1 μg ofeach sample was loaded on a NuPage 4-12% Bis-Tris gel (Invitrogen Cat.No. NP0322BOX) and run at 200 V for 30 minutes. Marker was Bio-RadPrecision Plus (Cat. No. 161-073). Bands corresponding to the predictedsizes of the heavy and light chains were observed with no evidence ofany contamination in any lane (data not shown).

ELISA Methodology

An ELISA was used to assay binding of humanized andhumanized/deimmunized anti-endoglin antibodies to endoglin. Briefly, anELISA was performed according to the following steps:

1. Coat a Nunc Maxisorp plate with MAB9811-01 (polyclonal anti-endoglinantibody) at 1500 ng/ml in PBS, 100 μl/well. Cover the plate with asealer and incubate overnight (16-24 hours) at 4° C.

2. Wash the plate 2× with −200 μl of PBS (without Tween).

3. Add 200 μl/well of BSA blocking solution (1% BSA) and incubate 60minutes at room temperature.

4. Wash the plate 3× with PBS containing Tween (PBS-T) using the BioTekplate washer.

5. Add 100 μl/well of CD105 (R&D Systems Cat 1097-EN) at 100 ng/ml inPBS-T with 0.1% BSA and incubate 60 minutes at room temperature.

6. Wash the plate 3× with PBS-T using the BioTek plate washer.

7. In test wells: add 100 μl/well of anti-endoglin antibodies at 20, 10,4, 2, 1, 0.5 and 0.2 ng/ml (diluted in PBS-T with 0.1% BSA) and incubate60 minutes at room temperature. In negative control wells: add 100μl/well of isotype matched control antibody.

8. Wash the plate 3× with PBS-T using the BioTek plate washer.

9. Add 100 μl/well of Goat anti-Human IgG conjugated to HRP (JacksonImmunoresearch), diluted 1:10000 in PBS-T with 0.1% BSA to all wells;incubate 30-60 minutes at room temperature.

10. Wash the plate 5× with PBS-T using the BioTek plate washer.

11. Add 100 μl/well of TMB substrate solution and incubate uncovered inthe dark for 15 minutes.

12. Stop the reaction by addition of 100 μl/well of TMB Stop Solution.

Samples are run in triplicate and the optical density is read toconstruct a standard curve and determine the binding constant.Statistical analysis is conducted using the Student's t-test or anotherstandard test.

Competition ELISA

Antibodies were tested in a competition ELISA for binding to CD105against biotinylated chimeric anti-CD105. Briefly, chimeric anti-CD105was biotinylated using a micro-biotinylation kit (Sigma, Catalog No.BTAG-1KT) following the manufacturer's instructions. Nunc ImmunoMaxiSorp 96-well flat-bottom microtiter plates were coated with mouseanti-human CD105 (Southern Biotechnologies, Catalog No. 9811-01) at 1.5μg/mL in phosphate buffered saline (PBS) overnight at 4° C. Thefollowing day, 100 ng/ml human CD105 (R&D Systems, Catalog No. 1097-EN)in PBS/2% BSA was added to the pre-coated plate and incubated at roomtemperature for 1 hour. Varying concentrations of humanized/deimmunizedanti-CD105 antibodies (4 μg/mL to 0.0018 μg/mL in three-fold dilutions)were mixed with a fixed concentration of biotinylated chimericanti-CD105 antibody (6.25 ng/ml) and added to the plate. Binding of thebiotinylated chimeric antibody was detected via streptavidin-HRP (Sigma,Catalog No. 55512) and TMB substrate (Sigma, Catalog No. T0440). OD450nm values were measured on a Dynex MRX TCII plate reader. The results ofthe competition analysis are illustrated in FIG. 6. Curves were fittedthrough the straight line portion of each of the plots of absorbanceagainst the log sample concentration and the equations of the lines wereused to calculate the concentrations of humanized orhumanized/deimmunized antibody required to inhibit biotinylated chimericantibody binding to CD105 by 50% (IC50). To allow for comparisons withinand between experiments, IC50 values of humanized orhumanized/deimmunized variants were normalized against the referenceantibody that was included on each plate to give a value for the folddifference. IC50 values are relative to chimeric anti-CD105 and arerepresentative of three experiments. Summary ELISA data are presented inTable 1 of U.S. Pat. No. 8,221,753 and include antibody expressionlevels (μg/ml) as assayed in saturated static cultures.

Table 1 of U.S. Pat. No. 8,221,753 provided the characteristics ofhumanized and humanized/deimmunized antibody variants. IC50 values arerelative to chimeric anti-endoglin antibody and are representative ofthree experiments. Antibody expression levels (μg/ml) were assayed insaturated static cultures. The level of deimmunization is represented byan arbitrary scale based upon the location in the epitopes of themutations. Accordingly, the present inventors have demonstrated thesuccessful preparation of humanized and deimmunized anti-endoglinantibodies.

Example 2 BIAcore (Surface Plasmon Resonance: SPR) Analysis of Humanizedand Deimmunized Anti-Endoglin Antibody Binding

Affinity of antibodies can be assessed using, for example, BIAcoreanalysis using standard protocols. Briefly, protein A is chemicallycoupled to a BIAcore CM5 chip, with the amount of protein A immobilizedcorresponding to 2000 RU. Subsequent steps are performed in a runningbuffer of 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% TWEEN, pH=7.4, at25 degrees Celsius using a 10 Hz data collection rate. Anti-endoglinantibody (10 nM) is captured at a 10 μL/min flow rate by the immobilizedprotein A on the BIAcore chip: typically, capture times of 20, 40 and 80seconds allow the capture of antibody densities corresponding to 130 RU,330 RU and 570 RU, respectively. Start-up cycles are performed usingrunning buffer at a flow rate of 40 μL/min, a contact time of 90 secondsand a dissociation time of 90 seconds. Sample cycles are performed usingrecombinant endoglin at concentrations ranging from 0 to 40 nM. Endoglinis passed over the BIAcore chip containing captured antibody at a flowrate of 40 μL/min with a contact time of 525 seconds and dissociationtime of 2500 seconds. Eight sample cycles are typically performed ateach antibody capture density. Regeneration of the chip is accomplishedusing 10 mM glycine pH=1.7. Data analysis is performed using BIAcoreT100 Evaluation Software v1.1 Signals generated using BIAcore chips withdifferent captured antibody densities are compared and data generated inthe absence of recombinant endoglin are used to adjust for theintraassay blank signal. For fitting of the data, the R_(max) is allowedto float to account for variation in capture levels of each antibody ineach cycle. Data from each capture density is fitted simultaneouslyduring analysis of each antibody. BIAcore data are presented in Table 2for chimeric, humanized and deimmunized anti-endoglin antibodies,including k_(a) (1/Ms), k_(d) (1/s), K_(D) (M) and Chi² (RU²).

TABLE 2 Anti-endoglin k_(a) k_(d) K_(D) Chi² Antibody (×10⁴) (×10⁻⁵)(×10⁻¹⁰) (×10⁻²) VK1AAVH1R 6.40 3.41 5.33 6.84 Chimeric 5.47 3.28 6.006.78 VK1AAVH1Q 3.56 3.03 8.50 7.07 VK1AAVH1A2 4.60 3.25 7.06 5.52 Table2. BIAcore binding data for chimeric anti-endoglin antibody andhumanized/deimmunized anti-endoglin antibodies VK1AAVH1R, VK1AAVH1Q andVK1AAVKVH1A2.

Example 3 Antibody Avidity and Number of Available Epitopes onEndoglin-Expressing Cells

Antibody avidity and number of available epitopes on endoglin-expressingcells can be assessed utilizing Scatchard plot analyses using standardprotocols.

Briefly, Scatchard plot analyses of direct binding of radiolabeledhumanized anti-endoglin antibodies to endoglin-expressing KM-3 leukemiacells and sub-confluent proliferating HUVECs are carried out. Thepurified anti-endoglin antibodies are individually radiolabeled with¹²⁵I using Iodo-Gen and according to standard methods known to thoseskilled in the art. The radiolabeled humanized anti-endoglin antibodiesare assayed for the mean number of iodine atoms per IgG molecule.Titration experiments are carried out using a fixed amount (0.1 μg) ofeach ¹²⁵I-labeled mAb and 2-fold serial increments ofendoglin-expressing HUVEC cells to determine antigen-binding activity.Analysis of Scatchard plot of binding data is carried out according toknown methods. An equilibrium constant and an average maximal number ofmAb bound/cell are estimated by this analysis.

Example 4 Identification of T-Cell Epitopes in Humanized Anti-EndoglinAntibodies

Sequences of humanized variable regions were tested by iTope™ analysis.Humanized variable region sequences were divided into overlapping9-15-mer peptides. The variable region sequences were analyzed forpromiscuous high affinity binding to human WIC class II (potential Tcell epitopes) using iTope™, an in silico analytic tool that determinesthe affinity of peptides for WIC Class II by computational analysis.Sequences with the lowest frequencies of potential T cell epitopes fromthe iTope™ analysis are identified as leads for generation of ahumanized antibody. The selected humanized variable region sequences mayredesigned through inclusion of mutations in order to remove potential Tcell epitopes. Mutations are designed using iTope™ to reduce oreliminate MHC class II binding. Alternatively, germ-line human sequencescan be substituted at sites of potential T cell epitopes or alternativesequences may be substituted.

FIGS. 19-23 of U.S. Pat. No. 8,221,753, which is hereby incorporated byreference is its entirety, present the predicted binding of 9 merpeptides for the humanized anti-endoglin antibody containing the lightchain HuVK_v0 and the heavy chain HuVH_v0, noted in FIG. 3B of thepresent application.

Example 5 Design of Anti-CD105 Humanized/Deimmunized Antibodies

This example describes the design of therapeutic monoclonal,humanized-deimmunized antibodies targeting human CD105 that exhibitreduced immunogenicity.

The promiscuous high affinity WIC class II binding sequences identifiedusing iTope™ (data not shown) were further analyzed by iTope™ in orderto identify amino acid substitutions at key MHC class II pocketpositions that would reduce or eliminate peptide binding to MHC classII. Since all the sequences overlapped CDRs, consideration was alsogiven to the CDR location of the changes (potential antigen contactresidues) and the physicochemical characteristics of the original andreplacement amino acids. TCR contact residues and residues outside themain binding groove involved in the stabilization of peptide/WIC classII-TCR interactions were also considered for replacement.

In VHV1, a 9-mer peptide lying completely within CDR2 and starting atresidue 51 was identified as a promiscuous high affinity MEW class IIbinding peptide. The most successful method for elimination of WIC classII binding is to target the first amino acid of the 9-mer (the pocket 1or p1 position) where removal of the hydrophobic side-chain orreplacement with a hydrophilic side-chain eliminates MHC class IIbinding. However, this type of radical amino acid replacement may notalways be successful in retaining antibody affinity, hence secondarypocket positions (p4, p6, p7 or p9), alone or in combination, were alsoassessed. iTope™ analysis revealed that targeting the p4 position ofthis peptide by changing K52b to Q or R is predicted to significantlyreduce MHC class II binding while replacement of 151 at p1 with A ispredicted to remove binding entirely (See, Table 5 of U.S. Pat. No.8,221,753).

Crystal structures of antibody/antigen complexes suggest that 151 mayinfrequently contact with antigen; however a radical change of I to A (asubstitution for disrupting a p1 anchor position) at this position couldaffect the overall conformation of the CDR. Therefore, relativelyconservative changes at K52b (p4 anchor position) were also includedsince this residue is solvent exposed but may not contact antigen.Finally, additional mutations lying outside the CDR were also designed(G49 to A or S) to assess the destabilizing effect on peptide/MHC classII/TCR interactions. Table 6 of U.S. Pat. No. 8,221,753 lists thehumanized/deimmunized variant VH regions that were constructed; the SEQID NOS for the corresponding nucleotide and amino acid sequences areindicated next to the constructs.

Two promiscuous high affinity MEW class II binding peptides wereidentified in VKV2 and VKV1. The first, with a p1 anchor at V19,partially overlaps CDR1 and the second, with a p1 anchor at 148,overlaps CDR2. Both p1 anchors lie outside the CDRs and were targeted bymutation to A, which may completely remove MHC class II binding (Table7). However, both these residues may be involved in the maintenance ofthe conformations of CDRs 1 and 2; therefore, additional mutations weredesigned that significantly reduced MHC class II binding (Table 7). Inboth cases, p4 residues were targeted by mutation of T to S. T22S alsolies outside the CDR and is less likely to affect CDR conformation thanV19A. T51 lies inside CDR2; however evidence from crystal structures ofantibodies complexed with antigen suggests that this residue rarelycontacts antigen. Table 6 of U.S. Pat. No. 8,221,753 lists humanized andhumanized/deimmunized VK regions that were constructed.

Example 6

This example describes a method of screening anti-endoglin antibodiesfor T-cell epitopes. The interaction between MHC, polypeptide and T cellreceptor (TCR) provides the structural basis for the antigen specificityof T cell recognition. T cell proliferation assays test the binding ofpolypeptides processed from antibodies to MHC and the recognition ofMHC/polypeptide complexes by the TCR. In vitro T cell proliferationassays of the present example, involve the stimulation of peripheralblood mononuclear cells (PBMCs), containing antigen presenting cells(APCs) and T cells. Stimulation is conducted in vitro using intactanti-endoglin antibodies. Stimulated T cell proliferation is measuredusing ³H-thymidine (³H-Thy) and the presence of incorporated³H-Thymidine is assessed using scintillation counting of washed fixedcells.

All humanized and humanized/deimmunized VH and VK region genes weresynthesized using a series of overlapping oligonucleotides that wereannealed, ligated and PCR amplified to give full length synthetic Vregions. The assembled variants were then cloned directly into AntitopeLtd.'s pANT expression vector system for IgG1 heavy chains and kappalight chains.

Purification of Antibodies

Anti-endoglin antibodies were purified from the supernatants ofmammalian cultures by protein A chromatography. Buffer exchange andprotein concentration was done using PBS pH=7.4. Anti-endoglin antibodywas further purified by size exclusion chromatography using a SephacrylS200 column (GE Healthcare, AMersham, UK). The major peak is collected,filter sterilized and shown to have endotoxin levels <5 EU/mg using anEndosafe-PTS (Charles River, Margate, UK). The purified antibodies arestored at 4 degrees Celsius. Final concentrations were determined by UVabsorption using calculated molar extinction coefficients, where A2801.0=1.62 mg/mL. Each antibody was then diluted to 100 μg/mL in AIMVculture medium.

Preparation and Selection of Donor PBMC

Peripheral Blood Mononuclear cells (PBMC) are isolated from healthycommunity donor buffy coats (from blood drawn within 24 hours) which areobtained from the UK National Blood Transfusion Service (Addenbrooke'sHospital, Cambridge, UK) according to approval granted by Addenbrooke'sHospital Local Research Ethics Committee. PBMC are isolated from buffycoats by Lymphoprep (Axis-shield, Dundee, Scotland) densitycentrifugation and CD8+ T cells are depleted using CD8+ RossetteSep™(StemCell Technologies, Inc.). Donors are characterized by identifyingHLA-DR haplotypes using a Biotest HLA SSP-PCR based tissue-typing kit(Biotest, Landsteinerstralβe, Denmark). T cell responses to a controlantigen, Keyhole Limpet Haemocyanin (KLH) (Pierce, Rockford, Ill., USA)are determined for a positive control. PBMC were then frozen and storedin liquid nitrogen until required. When required for use, cells arethawed rapidly in a water bath at 37° C. before transferring to 10 mlpre-warmed AIM V medium.

A cohort of 20 donors is selected to best represent the number andfrequency of HLA-DR allotypes expressed in the world population.Analysis of the allotypes expressed in the cohort against thoseexpressed in the world population revealed that coverage of >80% isachieved and that all major HLA-DR alleles (individual allotypes with afrequency >5% expressed in the world population) are well represented. Asummary of donor haplotypes is provided in FIG. 23, and a comparison ofthe frequency of donor allotypes used in the study versus those presentin the world population is made.

PBMCs from each donor are thawed, counted and viability assessed. Cellswere revived and resuspended in AIMV culture medium to 4-6×10⁶ PBMC/mL.For each donor, bulk cultures were established in which a total of 1 mLproliferation cell stock was added to a 24-well plate. A total of 1 mLof each diluted test sample was added to the PBMC to give a finalconcentration of 50 μg/mL per antibody sample. For each donor, apositive control (cells incubated with 100 μg/mL KLH) and a negativecontrol (cells incubated with culture media only) were also included.For the first 4 donors, an additional control was included to test formodulation of T cell responses by the test samples, where test sampleand KLH were added to the PBMC. Comparison of these samples with KLHalone can be used to assess the effects of the test samples onproliferation. Cultures were incubated for a total of 8 days at 37degrees Celsius with 5% carbon dioxide. On days 5, 6, 7 and 8, the cellsin each well are gently resuspended and three 100 μL aliquots aretransferred to individual wells of a round bottom 96 well plate. Thecultures are pulsed with 1 μCi ³[H]-Thy (Perkin Elmer, Waltham, Mass.)in 100 μL AIMV culture medium and incubated for a further 18 hoursbefore harvesting onto filter mats using a TomTec Mach III cellharvester. Counts per minute (cpm) for each well are determined byMeltilex™ (Perkin Elmer®, Waltham, Mass., USA) scintillation counting ona Microplate Beta Counter (Perkin Elmer®, Waltham, Mass., USA) inparalux, low background counting mode.

Results are expressed as stimulation indices, where the stimulationindex (SI) is derived by division of the proliferation score (e.g.,counts per minute of radioactivity) measured to the test anti-endoglinantibody by the score measured in cells not contacted with a testanti-endoglin antibody. All basal cpm for the control wells are abovethe minimum threshold for the assay of 150 cpm.

For proliferation assays, an empirical threshold of a stimulation index(SI) equal to or greater than 2 (SI≥2) has been previously establishedwhereby samples inducing proliferative responses above this thresholdare deemed positive (where included, borderline SIs≥1.90 arehighlighted). Extensive assay development and previous studies haveshown that this is the minimum signal to noise threshold allowingmaximum sensitivity without detecting large numbers of false positiveresponses. Positive responses are defined by the following statisticaland empirical thresholds:

1. Significance (p<0.05) of the response by comparing cpm of test wellsagainst medium control wells using unpaired two sample student's t-test.

2. Stimulation index greater than 2 (SI≥2), where SI=mean of test wells(cpm)/mean medium control wells (cpm).

In addition, intra-assay variation is assessed by calculating thecoefficient of variance and standard deviation (SD) of the raw data fromreplicate cultures.

Results for the EpiScreen time course proliferation assay with theanti-endoglin antibodies are shown in FIG. 24 of U.S. Pat. No. 8,221,753and summarized in tabular form (Table 8 of U.S. Pat. No. 8,221,753). Thechimeric antibody stimulated responses in 4 of 20 donors (20% of thestudy cohort) and, although two of the donor responses were borderline(1.92 and 1.95 for donors 11 and 17, respectively), they weresignificantly different from background (p<0.05). The humanized antibodyVK1VH1 stimulated responses in 2 of 20 donors (10% of the study cohort)including one borderline response (1.91 for donor 20) that wassignificantly different from background (p<0.05). It is noteworthy thatdonors 11 and 20 responded to both of these antibodies suggesting thatthere could be a shared T cell epitope. In contrast, none of the donorsin the study cohort responded positively to the deimmunizedanti-endoglin antibody VK1AA VH1A2. Results with the control antigen KLHshow that there was a good correlation between positive and negativeresults, indicating a high level of reproducibility in the assay.

Example 7

EpiScreen™ T Cell Epitope Mapping

EpiScreen™ is an ex vivo technology for measurement of T cell epitopesin whole antibodies or for mapping the sequence location of such T cellepitopes as described in more detail below.

EpiScreen™ Donor Selection

Peripheral Blood Mononuclear cells (PBMC) are isolated from healthycommunity donor buffy coats (from blood drawn within 24 hours) which areobtained from the UK National Blood Transfusion Service (Addenbrooke'sHospital, Cambridge, UK) according to approval granted by Addenbrooke'sHospital Local Research Ethics Committee. PBMC are isolated from buffycoats by Lymphoprep (Axis-shield, Dundee, Scotland) densitycentrifugation and CD8+ T cells are depleted using CD8+ RossetteSep™(StemCell Technologies, Inc.). Donors are characterized by identifyingHLA-DR haplotypes using a Biotest HLA SSP-PCR based tissue-typing kit(Biotest, Landsteinerstralβe, Denmark). T cell responses to a controlantigen, e.g., Keyhole Limpet Haemocyanin (KLH) (Pierce, Rockford, USA)are also determined for a positive control. A cohort of 54 donors isselected to best represent the number and frequency of HLA-DR allotypesexpressed in the world population. Analysis of the allotypes expressedin the cohort against those expressed in the world population revealedthat coverage of >80% is achieved and that all major HLA-DR alleles(individual allotypes with a frequency >5% expressed in the worldpopulation) are well represented. A summary of donor haplotypes isprovided, and a comparison of the frequency of donor allotypes used inthe study versus those present in the world population is made.

Donor details and haplotypes. Donor responses (SI) to KLH are tested intwo independent experiments. Test 1 is performed on freshly isolatedPBMC and an antibody is the re-test in the current study. Responses thatdid not produce the same result (i.e., positive or negative) in bothtests are highlighted. Donors with very low basal cpm (<150 cpm) areexcluded from the analysis.

EpiScreen Analysis: Proliferation Assays

EpiScreen™ is used to test overlapping peptides derived from thesequence of chimeric, humanized and humanized/deimmunized antibodies.Overlapping peptides are designed. A series of 128×15-mer peptidesoverlapping by 12 amino acids are synthesized together with 1×14-mer and1×11-mer and used to stimulate peripheral blood mononuclear cells (PBMC)derived from a cohort of 51 healthy donors using EpiScreen™ T cellepitope mapping. Individual peptides are tested in replicate culturesand responses are assessed using T cell proliferation assays to identifythe precise location of epitopes. PBMC from each donor are thawed,counted and assessed for viability. Cells are revived in roomtemperature AIM V culture medium (Invitrogen, Carlsbad, Calif.) beforeadjusting the cell density to 2.5×10⁶PBMC/ml (proliferation cell stock).Peptides are dissolved in DMSO (Sigma-Aldrich, St Louis, Mo., USA) to afinal concentration of 10 mM. Peptide culture stocks are then preparedby diluting into AIM V culture medium to a final concentration of 5 Foreach peptide and each donor, sextuplicate cultures are established byadding 100 μl of the peptide culture stocks to 100 μl of proliferationcell stock in a flat bottomed 96 well plate. Both positive and negativecontrol cultures are also established in sextuplicate. A total of 9×96well plates are used for each donor, and each plate is sufficient totest 15 peptides with one negative control (carrier alone) insextuplicate. On the final plate, a positive control is added.

Cultures are incubated for a total of 6 days before adding 0.5 μCi³[H]-Thymidine (Perkin Elmer®, Waltham, Mass., USA) to each well.Cultures are incubated for a further 18 hours before harvesting ontofilter mats using a TomTec Mach III cell harvester. Counts per minute(cpm) for each well are determined by Meltilex™ (Perkin Elmer®, Waltham,Mass., USA) scintillation counting on a Microplate Beta Counter (PerkinElmer®, Waltham, Mass., USA) in paralux, low background counting mode.

For proliferation assays, an empirical threshold of a stimulation index(SI) equal to or greater than 2 (SI≥2) has been previously establishedwhereby samples inducing proliferative responses above this thresholdare deemed positive (where included, borderline SIs≥1.90 arehighlighted). Extensive assay development and previous studies haveshown that this is the minimum signal to noise threshold allowingmaximum sensitivity without detecting large numbers of false positiveresponses. Positive responses are defined by the following statisticaland empirical thresholds:

1. Significance (p<0.05) of the response by comparing cpm of test wellsagainst medium control wells using unpaired two sample student's t-test.

2. Stimulation index greater than 2 (SI≥2), where SI=mean of test wells(cpm)/mean medium control wells (cpm).

In addition, intra-assay variation is assessed by calculating thecoefficient of variance and standard deviation (SD) of the raw data fromreplicate cultures.

Proliferation assays are set up in sextuplicate cultures (“non-adjusteddata”). To ensure that intra assay variability is low, data is alsoanalyzed after removing the maximum and minimum cpm values (“adjusteddata”) and the SI of donor responses are compared using both data sets.Details of donor SIs from both adjusted and non-adjusted data sets areprepared. T cell epitopes are identified by calculating the averagefrequency of responses to all peptides in the study +2×SD (backgroundresponse rate). Any peptide(s) that induced proliferation above thisthreshold is considered to contain a T cell epitope.

In Silico iTope™ Analysis of Peptides

The sequences of peptides that are positive in the proliferation assayare analyzed using Antitope's predictive iTope™ software. This softwarepredicts favorable interactions between amino acid side chains of thepeptide and specific binding pockets within the MEW class II bindinggroove. The location of key binding residues is determined by generating10-mer peptides that overlapped by one amino acid spanning the longpeptide sequence. Each 10-mer is tested against Antitope's database ofMHC class II allotypes and scored based on their fit and interactionswith the MEW class II molecules. Peptides that produced a high bindingscore against a large number of alleles are considered to contain thecore 9-mer.

Identification of T Cell Epitopes

All peptides identified using the EpiScreen™ Analysis described aboveare successfully synthesized for testing against 51 healthy donors (54donors are originally selected; donors may be excluded from the analysisdue to low basal cpm, i.e., below the cut off value of 150 cpm).Positive responses are defined by donors that produced a significant(p<0.05) response with a SI≥2 to any given peptide. Borderline responses(a significant (p<0.05) response with an SI≥1.90) are also included. Theoutputs from non-adjusted and adjusted data analyses are compared toensure that intra-assay variability is low and that positive responsesare not the result of spurious proliferation in individual wells. Theresults from each analysis showed little difference between the methods;consequently, the T cell epitope map is compiled using the adjusted dataanalysis. Donor stimulation indices from both non-adjusted and adjustedanalyses are prepared. T cell epitopes are identified by calculating theaverage frequency of the responses to all peptides in the study plustwice the standard deviation (termed ‘background response rate’). Thisis calculated to be 5.6% and is the equivalent of inducing a positiveresponse in three or more donors. Peptides inducing proliferativeresponses above this threshold are considered to contain a T cellepitope.

Immunogenicity Testing of Lead Variants Using EpiScreen™

Lead variants are purified and compared against the wild-typepolypeptide using EpiScreen™ time course T cell assays. A large numberof healthy donors representing the world population according toexpression of HLA allotypes are selected from a donor library asdescribed above. Donors are stimulated with each protein in separatebulk cultures containing 2-4×10⁶ CD8⁺ T cell depleted PBMC. Replicatesamples (of T blasts) are removed from bulk cultures on days 5-8, andproliferation along with IL-2 secretion (ELISPOT) is assessed. Tofurther validate the assessment between wild type and variants, thestudy cohort is supplemented with responding donors from the EpiScreen™T cell epitope mapping study (provided sufficient numbers of CD8⁺ T celldepleted PBMC remain).

In order to confirm loss of immunogenicity in lead variants, an analysisof T cell immunogenicity by EpiScreen™ time course T cell assays isundertaken as follows:

(i) Buffy coats from healthy donors (with >80% DRB 1 allotypic coveragefor world population) are used to isolate PBMC which containphysiological levels of APC and CD4+ T cells;

(ii) Each donor is tested against positive control antigens includingkeyhole limpet haemocyanin (a potent neoantigen);

(iii) CD8+ T cells are depleted to exclude the detection of MEW class Irestricted T cell responses;

(iv) Lead variants and wild-type polypeptides are compared against eachother to evaluate relative capacity to activate T cells CD4+ T cells;

(v) Data is analyzed using previously validated assay parameters withpositive responses of SI>2 supported by additional information includingstatistical and frequency analysis;

(vi) Data from EpiScreen™ time course T cell assays provides informationon the magnitude and kinetics of T cell responses to individualmolecules;

(vii) Any remaining PBMC from donors that produce positive responses isarchived and is available for use in repeat testing studies; and

(viii) An assessment is made of association between donor allotype andresponses to wild-type polypeptide and any responses to variant leads.

Example 8 In Vivo Efficacy Study of M1043 in a CarbonTetrachloride-Induced Liver Fibrosis Model

The purpose of this example is to examine the effects of M1043 Antibodythat binds to mouse endoglin to competitively inhibit mouse BMP bindingto mouse endoglin in a carbon tetrachloride-induced liver fibrosismodel.

M1043 heavy chain variable region (SEQ ID NO: 124)QVQLQQSGAELVKPGSSVKISCKASGYTFTSYDMHWIKQQPGNGLEWIGWIYPGNGNTKYNQKFNGKATLTADKSSSTAYMQLSSLTSEDSA VYFCARGKFGVGDYWGQGVMVTVSSM1043 light chain variable region (SEQ ID NO: 125)DTVLTQSPALAVSPGERVSISCRASEGVNSYMHWYQQKPGQQPKLLIYIASNLASGVPARFSGSGSGTDFTLTIDPVEADDTATYFCQQSWN DPYTFGAGTKLELKRMaterials And Methods

Test Substances

M1043 Antibody was provided by TRACON Pharmaceuticals, Inc.Isotype-matched antibody (rat IgG1) was purchased from Bio X cell (Cat#BE0088). Dosing solution was prepared by diluting the stock solution ofisotype-matched antibody or M1043 Antibody with vehicle (sterilizedPhosphate Buffered Saline (PBS)).

Animals

Seven-week-old female C57BL/6J mice (17˜21 g) were obtained from JapanSLC (Japan).

Animals were housed and fed with normal diet (CE-2; CLEA Japan, Japan)under conventional conditions. All animals used in this study were caredfor following guidelines;

1. Act on Welfare and Management of Animals (Ministry of theEnvironment, Act No. 105 of Oct. 1, 1973)

2. Standards Relating to the Care and Management of Laboratory Animalsand Relief of Pain (Notice No. 88 of the Ministry of the Environment,Apr. 28, 2006)

3. Guidelines for Proper Conduct of Animal Experiments (Science Councilof Japan, Jun. 1, 2006)

Environment

The animals were maintained in a specific pathogen-free (SPF) facilityunder controlled conditions of temperature (23±2° C.), humidity(45±10%), lighting (12-hour artificial light and dark cycles; light from8:00 to 20:00) and air exchange. A high pressure was maintained in theexperimental room to prevent contamination of the facility.

Animal Housing

The animals were housed in polycarbonate cages KN-600 (NatsumeSeisakusho, Japan) with a maximum of 4 mice per cage. SterilizedPaper-Clean (Japan SLC) was used for bedding and replaced once a week.

Food and Drink

Sterilized normal diet was provided ad libitum, being placed in a metallid on the top of the cage. Distilled water was also provided ad libitumfrom a water bottle equipped with a rubber stopper and a sipper tube.Water bottles were replaced once weekly, cleaned, sterilized in anautoclave and reused.

Animal and Cage Identification

Mice were identified by numbers engraved on earrings. Each cage was alsogiven a specific identification code.

Randomization

Mice were divided into 3 groups of 8 mice based on their body weight onthe day before the start of the treatment (day 13).

Induction of CCl₄-Induced Liver Fibrosis Model

Mice were intraperitoneally administered 5% CCl₄ (Sigma-Aldrich, USA) inmineral oil (Sigma-Aldrich) in a volume of 100 μL twice a week (day 0,4, 7, 11, 14, 18, 21 and 25) after acclimation.

Routes of Drug Administration

Isotype-matched antibody and M1043 Antibody were administered byintravenously route in a volume of 5 mL/kg.

Treatment Dose

Isotype-matched antibody and M1043 Antibody were administered to themice twice weekly at a dose of 10 mg/kg twice weekly.

Individual body weight was measured daily. Survival, clinical signs andbehavior of mice was also monitored daily.

Plasma Specimen Collection

The blood samples were collected in polypropylene tubes withanticoagulant (Novo-Heparin; Mochida Pharmaceutical, Japan) andcentrifuged at 1,000×g for 15 minutes at 4° C. The supernatant wascollected and stored at −80° C.

Tissue Specimen Collection

Fresh and frozen liver specimens were collected using conventionaltechniques.

Histological Analyses

To visualize collagen deposition, sections were cut from paraffin blocksof left lateral liver tissue prefixed in Bouin's solution and stainedwith picro-Sirius red solution (Waldeck, Germany). For quantitativeanalysis of fibrosis area, bright field images of Sirius red-stainedsection were randomly captured using a digital camera (DFC280; LeicaMicrosystems, Germany) at 100-fold magnification, and the Siriusred-stained positive areas in 5 field/section were measured using ImageJsoftware (National Institute of Health, USA). The quantification wasdone in a blinded fashion.

Statistical Tests

Statistical analyses were performed using Bonferroni Multiple comparisontest on GraphPad Prism 4 (GraphPad Software, USA). P values<0.05 wereconsidered statistically significant. Results were expressed as mean±SD.

Experimental Design and Treatment

Treatment groups were separated as follows:

Group 1: Disease-Control

Eight CCl₄-induced liver fibrosis model mice were kept without anytreatment until day 28.

Group 2: Isotype Matched Antibody

Eight CCl₄-induced liver fibrosis model mice were intravenouslyadministered vehicle supplemented with isotype-matched antibody at adose of 10 mg/kg twice weekly at day 14, 17, 21 and 24.

Group 3: Antibody A

Eight CCl4-induced liver fibrosis model mice were intravenouslyadministered vehicle supplemented with M1043 Antibody at a dose of 10mg/kg twice weekly at day 14, 17, 21 and 24. All animals were sacrificedon day 28.

Table 3 summarizes the treatment schedule:

No. Dose Volume Group mice Test substance (mg/kg) (mL/kg) Regimen 1 8Disease Control 0 — — 2 8 Isotype-Matched 10 5 IV, twice weekly,Antibody Day 14, 17, 21 and 24 3 8 M1043 Antibody 10 5 IV, twice weekly,Day 14, 17, 21 and 24

Animal Monitoring and Sacrifice

The viability, clinical signs and behavior were monitored every day.Body weight was recorded daily during the treatment period. Animals weresacrificed by exsanguination through the heart puncture under etheranesthesia (Wako Pure Chemical Industries, Japan).

Results

Body weight changes and general condition (FIG. 10)

There were no significant differences either between the isotype-matchedantibody and disease control groups or between isotype-matched antibodyand M1043 Antibody groups.

In the present study, none of the animals showed deterioration ingeneral condition.

Body Weight on the Day of Sacrifice (FIG. 11A and Table 4)

There were no significant differences either between the Isotype-matchedantibody and disease control groups or between isotype-matched antibodyand M1043 Antibody groups.

Liver Weight (FIG. 11B and Table 4)

The isotype-matched antibody group showed a significant decrease in meanliver weight compared with the disease-control group. There was nosignificant difference in mean body weight between the isotype-matchedantibody group and the M1043 Antibody group.

TABLE 4 Body weight and liver weight lsotype-Matched ParameterDisease-control Antibody M1043 Antibody (mean ± SD) (n = 8) (n = 8) (n =8) Body weight (g)  20.9 ± 0.7 20.6 ± 0.8 20.5 ± 0.7 Liver weight (mg)1058 ± 57 877 ± 96 896 ± 48

Histological Analyses

Sirius red staining (FIGS. 12A-F, FIG. 13 and Table 5) was conductedusing the protocol described above.

Extensive collagen deposition and bridging fibrosis were evident in theliver sections from the isotype-matched antibody and disease controlgroups. M1043 Antibody group showed a lower collagen deposition withless frequent formation of bridging fibrosis than the isotype-matchedantibody and disease control groups.

The percentage of fibrosis area (Sirius red-positive area) significantlydecreased in the M1043 Antibody group compared with the isotype-matchedantibody group (P<0.05).

TABLE 5 Fibrosis area Isotype-Matched Parameter Disease control AntibodyM1043 Antibody (mean ± SD) (n = 8) (n = 8) (n = 8) Sirius red-positive2.66 ± 0.61 2.70 ± 0.36 2.07 ± 0.52 area (%)Conclusion

In the present study, Antibody M1043 treatment showed a significantreduction of collagen deposition as evidenced by Sirius red positivearea. Furthermore, no abnormal findings were observed in the antibodytreatment group. These results suggested that M1043 Antibody treatmenthas anti-fibrosis efficacy without significant side effects.

Example 9 In Vivo Efficacy Study of M1043 and TRC105 inBleomycin-Induced Pulmonary Fibrosis

The purpose of this study was to examine the effects of M1043 thatcompetitively inhibits mouse BMP binding to mouse endoglin and antibodyTRC105 that binds mouse endoglin but does not inhibit mouse BMP bindingto mouse endoglin in a bleomycin-(BLM)-induced model of pulmonaryfibrosis.

Protocol

Pathogen-free 7 weeks old female C57BL/6J mice were obtained from JapanSLC, Inc. (Japan). At day 0, 60 mice were induced to develop pulmonaryfibrosis by a single intratracheal administration of bleomycin sulphate(BLM, Nippon Kayaku, Japan) in saline at a dose of 3 mg/kg, in a volumeof 50 μL per animal using Microsprayer® (Penn-Century, USA).

BLM-induced pulmonary fibrosis model mice were randomized into 4 groupsof 12 mice based on the body weight on the day before the start oftreatment.

Individual body weight were measured daily during the experimentalperiod.

Survival, clinical signs and behavior of mice were monitored daily.

Groups:

Group 1 (Disease-control): Twelve BLM-induced pulmonary fibrosis modelmice were kept without any treatment until day 20.

Group 2 (Isotype-Matched Antibody): Twelve BLM-induced pulmonaryfibrosis model mice were intravenously administered vehicle (Phosphatebuffered saline) supplemented with isotype-matched antibody [rat IgG1from BioExcel catalog BE0088 (25 mg)] at a dose of 10 mg/kg twice weeklyat day 7, 10, 14 and 17.

Group 3 (M1043 Antibody): Twelve BLM-induced pulmonary fibrosis modelmice were intravenously administered vehicle supplemented with M1043 ata dose of 10 mg/kg twice weekly at day 7, 10, 14 and 17.

Group 4 (TRC105 human/mouse chimeric antibody): Twelve BLM-inducedpulmonary fibrosis model mice were intravenously administered vehiclesupplemented with TRC105 at a dose of 10 mg/kg twice weekly at day 7,10, 14 and 17.

Group 5 (BIBF1120 inhibitor for Idiopathic Pulmonary Fibrosis—positivecontrol, Boehringer Ingelheim): Twelve BLM-induced pulmonary fibrosismodel mice were orally administered 1% CMC supplemented with BIBF1120 ata dose of 100 mg/kg once daily from day 7 to 21.

Mice in all groups were terminated for the following assays at day 21.

Sample Collection and Results

Biochemical analysis (by examiners unaware of treatment assignment);lung hydroxyproline was quantified by a hydrolysis method. See, FIG.14A.

Histological analysis of lung sections was conducted by practitionerswho are not aware of the Group identity. Masson's Trichrome staining isconducted and an Ashcroft Score estimate was determined. See, FIG. 14B.

There were no significant differences in body weight between the fivetreatment groups by Bonferroni Multiple Comparison Testing (p>0.05comparing the isotype control antibody group versus each other group).See, FIG. 14C.

There were no significant differences in survival between the fivetreatment groups by Bonferroni Multiple Comparison Testing (p>0.05comparing the isotype control antibody group versus each other group).See, FIG. 14D.

Ashcroft scores are presented in FIG. 14E. There was no significantdifference in Ashcroft score between the isotype-control antibody groupand the disease control group. There was no significant difference inAshcroft score between the isotype-control antibody group and the M1043treated group. Ashcroft score in the TRC105 antibody treated group wassignificantly lower than the isotype-control antibody group. Ashcroftscore in the BIBF1120 treated group was significantly lower than theisotype-control antibody group Statistical tests were performed usingBonferroni Multiple Comparison Test. P values<0.05 were consideredstatistically significant.

Example 10 In Vivo Efficacy Study of M1043, TRC105 and TRC205 in a Modelof Non-Alcoholic Steatohepatitis (NASH)

The purpose of this study was to examine the protective effects ofendoglin antibodies on a murine model of NASH. Test articles includedM1043, a rat IgG1 antibody that competitively inhibits mouse BMP bindingto mouse endoglin; TRC105, a human murine chimeric IgG1 antibody thatbinds murine endoglin to cause endoglin to be shed into plasma where itmay bind BMP and TGF-beta, but does not directly inhibit murine BMPbinding to murine endoglin; and TRC205, a human deimmunized IgG4antibody that binds murine endoglin to cause endoglin to be shed intoplasma where it may bind BMP and TGF-beta, but does not directly inhibitmurine BMP binding to murine endoglin.

Protocol

Pathogen-free 14-day-pregnant C57BL/6 mice were obtained from Japan SLC,Inc. (Japan).

NASH was established by a single subcutaneous injection of STZ (Sigma,USA) after birth and feeding with a high fat diet (HFD; CLEA Japan,Japan) ad libitum after 4 weeks of age (day 28±2).

Randomization of mice into 6 groups of 10 mice was done at 9 weeks ofage (day 63±2), the day before the start of treatment.

Individual body weight was measured daily during the treatment period.

Survival, clinical signs and behavior of mice were monitored daily.

Groups:

Group 1 (non-Diseased control; Normal-Vehicle Group): Ten mice were fedad libitum and be intravenously administered vehicle (Phosphate bufferedsaline) twice weekly at day 63, 66, 70, 73, 77, 80

Group 2 (Disease-control; NASH-Vehicle Group): Ten NASH mice were fedwith HFD ad libitum and intravenously administered vehicle (Phosphatebuffered saline) twice weekly at day 63, 66, 70, 73, 77 and 80.

Group 3 (Positive control; Temlisartan Group): Ten NASH mice were beorally administered pure water supplemented with telmisartan at a doseof 10 mg/kg once daily from day 63 to 84.

Group 4 (Antibody M1043: M1043 Group): Ten NASH mice were intravenouslyadministered vehicle supplemented with Antibody M1043 at a dose of 10mg/kg twice weekly at day 63, 66, 70, 73, 77 and 80.

Group 5 (Antibody TRC105; TRC105 Group): Ten NASH mice wereintravenously administered vehicle supplemented with Antibody TRC105 ata dose of 10 mg/kg twice weekly at day 63, 66, 70, 73, 77 and 80.

Group 6 (Antibody TRC205; TRC205 Group): Ten NASH mice wereintravenously administered vehicle supplemented with Antibody TRC205 ata dose of 10 mg/kg twice weekly at days 63, 66, 70, 73, 77 and 80.

Mice in all groups were terminated for the following assays at 12 weeksof age.

Sample Collection and Analysis

H&E staining of liver tissue was conducted to estimate NAFLD Activityscore.

TABLE 6 Components of NAFLD Activity Score (NAS) and Fibrosis StagingItem Score Extent Definition and Comment NAS Components Steatosis 0  <5%Refers to amount of surface area involved by steatosis as evaluated onlow to medium power examination; minimal steatosis (<5%) receives ascore of 0 to avoid giving excess weight to biopsies with very littlefatty change 1    5-33% 2 >33-66% 3 >66% Lobular 0 No foci Acidophilbodies are not Inflammation included in this assessment, nor is portalinflammation 1 foci/200x 2 2-4 foci/200x 3 foci/200x Hepatocyte 0 NoneBallooning 1 Few balloon cells The term “few” means rare but definiteballooned hepatocytes as well as cases that are diagnosticallyborderline 2 Many Most cases with prominent cells/prominent ballooningalso had Mallory's ballooning hyalin, but Mallory's hyaline is notscored separately for the NAS Fibrosis Stage (Evaluated separately fromNAS) Fibrosis 0 None 1 Perisinusoidal or periportal 1A Mild, zone 3,“delicate” fibrosis perisinusoidal 1B Moderate, zone 3, “dense” fibrosisperisinusoidal 1C Portal/periportal This category is included toaccommodate cases with portal and/or periportal fibrosis withoutaccompanying pericellular/perisinusoidal fibrosis 2 Perisinusoidal andportal/periportal 3 Bridging fibrosis 4 Cirrhosis

Total NAS score represents the sum of scores for steatosis, lobularinflammation, and ballooning, and ranges from 0-8. Diagnosis of NASH(or, alternatively, fatty liver not diagnostic of NASH) should be madefirst, then NAS is used to grade activity. In the reference study, NASscores of 0-2 occurred in cases largely considered not diagnostic ofNASH, scores of 3-4 were evenly divided among those considered notdiagnostic, borderline, or positive for NASH. Scores of 5-8 occurred incases that were largely considered diagnostic of NASH.

NAFLD scores are presented in Table 7.

TABLE 7 NAFLD Activity score Score Lobular Hepatocyte Steatosisinflammation ballooning NAS Group n 0 1 2 3 0 1 2 3 0 1 2 (mean ± SD)Normal-Vehicle 10 10  — — — 10  — — — 10  — — 0.0 ± 0.0 NASH-Vehicle 9 —6 3 — — 1 6 2 — — 9 5.4 ± 0.5 Telmisartan 9 — 9 — — 3 4 2 — 1 3 5 3.3 ±1.2 TRC105 8 1 6 1 — — 1 5 2 3 — 5 4.4 ± 1.4 TRC205 9 — 6 3 — 5 — 4 — 14 4 3.6 ± 1.1 M1043 8 — 6 2 — 1 1 6 — 2 2 4 4.1 ± 1.0 Item Score ExtentSteatosis 0  <5% 1  5-33% 2 >33-66% 3 >66% Lobular Inflammation 0 NoFoci 1  <2 foci/200x 2 2-4 foci/200x 3  >4 foci/200x HepatocyteBallooning 0 None 1 Few balloon cells 2 Many cells/prominent ballooning

Liver sections from the NASH-Vehicle group exhibited severe micro- andmacrovesicular fat deposition, hepatocellular ballooning andinflammatory cell infiltration. The NASH-Vehicle group showed asignificant increase in NAS compared with the Normal-Vehicle group. TheTelmisartan (positive control group), TRC205 and M1043 groups showedsignificant decreases in NAS compared with the NASH-Vehicle group. TheTRC105 group tended to decrease in NAS compared with the NASH-Vehiclegroup.

F4/80 is a mature macrophage marker. Liver sections from theNASH-Vehicle group showed an increased number and size of F4/80-positivecells in the liver lobule compared with the Normal-Vehicle group. LowerF4/80-positive signals and positive cells were observed in the TRC105,TRC205 and M1043 groups compared with the NASH-Vehicle group. No obviouschanges were observed in the Telmisartan group compared with theNASH-Vehicle group.

Sirius-Red Staining

Sirius-red staining was conducted to estimate the percentage of fibrosisarea. Liver sections from the NASH-Vehicle group exhibited collagendeposition in the pericentral region of liver lobule. The NASH-Vehiclegroup showed a significant increase in Sirius red-positive area comparedwith the Normal-Vehicle group. The Telmisartan group showed asignificant decrease in Sirius red-positive area compared with theNASH-Vehicle group. There were no significant differences in Siriusred-positive area between the NASH-Vehicle group and any of thetreatment groups. See, e.g., Table 8.

TABLE 8 Histology analysis Normal- NASH- Parameter Vehicle VehicleTelmisartan TRC105 TRC205 M1043 (mean ± SD) (n = 10) (n = 9) (n = 9) (n= 8) (n = 9) (n = 8) Sirius red-positive 0.30 ± 0.06 0.98 ± 0.21 0.67 ±0.12 0.92 ± 0.20 0.93 ± 0.14 0.86 ± 0.30 area (%)

Biochemistry

Several serum proteins were quantified to assess the damage to liverparenchyma (Table 9).

TABLE 9 Biochemistry Normal- NASH- Parameter Vehicle Vehicle TelmisartanTRC105 TRC205 M1043 (mean ± SD) (n = 10) (n = 9) (n = 9) (n = 8) (n = 9)(n = 8) Serum ALT (U/L) 15 ± 3  48 ± 21 29 ± 15 62 ± 57 28 ± 16 24 ± 4 Serum AST (U/L) 61 ± 21 126 ± 52  113 ± 32  204 ± 205 83 ± 41 76 ± 24Serum ALP (U/L) 339 ± 62  420 ± 121 444 ± 134 291 ± 119 264 ± 63  358 ±129 Liver triglyceride 5.9 ± 1.0 44.2 ± 6.9  23.5 ± 10.6 31.1 ± 20.329.3 ± 9.4  22.4 ± 5.4  (mg/g liver)

The NASH-Vehicle group showed a significant increase in serum alanineaminotransferase (ALT) levels compared with the Normal-Vehicle group.The Telmisartan, TRC205 and M1043 groups tended to decrease in serumalanine aminotransferase levels compared with the NASH-Vehicle group.There was no significant difference in serum alanine aminotransferaselevels between the NASH-Vehicle group and the TRC105 group.

The NASH-Vehicle group tended to increase in serum aspartateaminotransferase (AST) levels compared with the Normal-Vehicle group.The TRC205 and M1043 groups tended to decrease in serum aspartateaminotransferase levels compared with the NASH-Vehicle group. There wereno significant differences in serum aspartate aminotransferase levelsbetween the NASH-Vehicle group and any of the other groups.

The TRC205 group showed a significant decrease in serum alkalinephosphatase (ALP) levels compared with the NASH-Vehicle group. TheTRC105 group tended to decrease in serum alkaline phosphatase levelscompared with the NASH-Vehicle group. There were no significantdifferences in serum alkaline phosphatase levels between theNASH-Vehicle group and any of the other groups.

The NASH-Vehicle group showed a significant increase in livertriglyceride contents compared with the Normal-Vehicle group. TheTelmisartan, TRC205 and M1043 groups showed significant decreases inliver triglyceride contents compared with the NASH-Vehicle group. TheTRC105 group tended to decrease in liver triglyceride contents comparedwith the NASH-Vehicle group.

Gene Expression

Gene expression of several genes implicated in liver injury and fibrosiswere assessed. See, e.g., Table 10.

TABLE 10 Gene expression analyses Normal- NASH- Parameter VehicleVehicle Telmisartan TRC105 TRC205 M1043 (mean ± SD) (n = 10) (n = 9) (n= 9) (n = 8) (n = 9) (n = 8) TNF-α 1.0 ± 0.5 4.1 ± 1.6 3.2 ± 2.3 12.9 ±9.5  6.2 ± 2.4 4.4 ± 1.9 MCP-1 1.0 ± 0.5 4.7 ± 2.8 1.5 ± 1.1 5.7 ± 4.29.3 ± 9.1 3.2 ± 2.4 Alpha-SMA 1.0 ± 0.3 2.2 ± 1.1 0.7 ± 0.3 1.7 ± 0.71.1 ± 0.3 1.7 ± 0.4 TIMP-1 1.0 ± 0.2 8.9 ± 3.9 5.8 ± 3.7 25.4 ± 23.0 6.0± 2.6 7.6 ± 3.3

Alpha-smooth muscle actin mRNA expression levels was significantlyup-regulated in the NASH-Vehicle group compared with the Normal-Vehiclegroup. Alpha-smooth muscle actin mRNA expression levels wassignificantly down-regulated in the Telmisartan and TRC205 groupscompared with the NASH-Vehicle group. There were no significantdifferences in Alpha-smooth muscle actin mRNA expression levels betweenthe NASH-Vehicle group and any of the treatment groups.

Tissue inhibitor of metalloproteinase-1 mRNA expression levels tended toincrease in the NASH-Vehicle group compared with the Normal-Vehiclegroup. Tissue inhibitor of metalloproteinase-1 mRNA expression levelstended to decrease in the TRC205 group compared with the NASH-Vehiclegroup. Tissue inhibitor of metalloproteinase-1 mRNA expression levelswas significantly up-regulated in the TRC105 group compared with theNASH-Vehicle group. There were no significant differences in Tissueinhibitor of metalloproteinase-1 mRNA expression levels between theNASH-Vehicle group and any of the other groups.

Tumor necrosis factor-alpha mRNA expression levels tended to increase inthe NASH-Vehicle group compared with the Normal-Vehicle group. Tumornecrosis factor-alpha mRNA expression levels tended to increase in theTRC205 group compared with the NASH-Vehicle group. Tumor necrosisfactor-alpha mRNA expression levels was significantly up-regulated inthe TRC105 group compared with the NASH-Vehicle group. There were nosignificant differences in Tumor necrosis factor-alpha mRNA expressionlevels between the NASH-Vehicle group and any of the other groups.

Macrophage chemoattractant protein-1 mRNA expression levels tended toincrease in the NASH-Vehicle group compared with the Normal-Vehiclegroup. Macrophage chemoattractant protein-1 mRNA expression levelstended to decrease in the Telmisartan group compared with theNASH-Vehicle group. There were no significant differences in macrophagechemoattractant protein-1 mRNA expression levels between theNASH-Vehicle group and any of the other groups.

Liver Weight

The NASH-Vehicle group showed a significant increase in mean liverweight compared with the Normal-Vehicle group. The Telmisartan groupshowed a significant decrease in mean liver weight compared with theNASH-Vehicle group. The M1043 group tended to decrease in mean liverweight compared with the NASH-Vehicle group. There were no significantdifferences in mean liver weight between the NASH-Vehicle group and anyof the other groups.

The NASH-Vehicle group showed a significant increase in meanliver-to-body weight ratio compared to the Normal-Vehicle group. TheTelmisartan and M1043 groups showed significant decreases in meanliver-to-body weight ratio compared with the NASH-Vehicle group. TheTRC205 group tended to decrease in mean liver-to-body weight ratiocompared with the NASH-Vehicle group. There was no significantdifference in mean liver-to-body weight ratio between the NASH-Vehiclegroup and the TRC205 group.

Statistical Tests

Statistical tests were performed using Bonferroni Multiple ComparisonTest. P values<0.05 were considered statistically significant.

In the study, the improvement in serum ALT and AST levels indicate thatTRC205 and M1043 ameliorated hepatocellular injury and inflammation.TRC205 and M1043 groups showed reductions in inflammatory cellinfiltration in the liver, which resulted in the decrease of the lobularinflammation score of NAS and F4/80 immunostaining. Together theseresults suggest that TRC205 and M1043 possess hepatoprotective andanti-inflammatory properties against NASH. Further, of particular noteis the significant reduction of total NAS in the TRC205 and M1043groups. NAS is one of the clinical endpoints for assessing the activityof NASH, and is the key preclinical endpoint in the study. TRC205 andM1043 showed significant reductions of NAS.

Example 11

An ELISA was established to determine the binding of M1043 antibody tomouse endoglin as follows: Nunc Immuno MaxiSorp 96 well flat bottomplates (Fisher, Cat. No. DIS-971-030J) were coated with mouse endoglin(R&D Systems, Cat. No. 1320-EN-025) at 1 μg/ml in PBS overnight at 4° C.The following day the plate was washed three times with PBST beforeblocking with PBST/3% Marvel at room temperature for 1 hour. Afterblocking, the plate was washed three times with PBST, and varyingconcentrations (10 μg/ml to 0.014 μg/ml in three-fold serial dilutions)of either M1043 or an irrelevant rat IgG1 (Affymetrix, Cat. No. 14-4301)in PBST/3% Marvel were added to the plate and incubated for 1 hour atroom temperature. Binding of the antibodies was detected via anti-ratIgG peroxidase (Sigma, Cat. No. A9037) and TMB single solution substrate(Invitrogen, Cat. No. 00-2023). The reaction was stopped by adding 3 MHCl and OD450 nm values were measured on a Dynex MRX TCII plate reader.The results of the binding ELISA are shown in FIG. 15. The results showthat the M1043 antibody binds specifically to mouse endoglin since nobinding was observed with the isotype matched irrelevant antibody.

The ability of mouse BMP9 to compete with the rat antibody M1043 forbinding to mouse endoglin was determined by competition ELISA asfollows: 200 μg of M1043 antibody was biotinylated with a Lightning-Linkbiotinylation kit (Innova Biosciences, Cat. No. 704-0010) according tothe manufacturers recommendations. Nunc Immuno MaxiSorp 96 well flatbottom plates (Fisher Cat. No. DIS-971-030J) were coated with mouseendoglin (R&D Systems, Cat. No. 1320-EN-025) at 1 μg/ml in PBS overnightat 4° C. The following day the plate was washed three times with PBSTbefore blocking with PBST/3% Marvel at room temperature for 1 hour. Athreefold dilution series (30 μg/ml to 0.004 μg/ml) of mouse BMP9 (R&DSystems, Cat. No. 5566-BP-010) or an irrelevant rat IgG1 was mixed witha fixed concentration of biotinylated M1043 (40 ng/ml), added to theplate and incubated for 1 hour at room temperature. Binding of thebiotinylated antibody was detected via streptavidin-HRP (Sigma, Cat. No.55512) and TMB substrate (Sigma, Cat. No. T0440). OD450 nm values weremeasured on a Dynex MRX TCII plate reader. The results of thiscompetition ELISA are shown in FIG. 16. The results show that mouse BMP9competed with biotinylated M1043 for binding to mouse endoglin whereasan irrelevant molecule did not.

Example 12

An ELISA was established to determine the binding of biotinylated TRC205antibody to human CD105 as follows: The TRC205 antibody (SEQ ID NO89/SEQ ID NO 93; IgG4) was biotinylated using a Lightning-Link BiotinConjugation Kit (Type A) (Innova Biosciences Cat. No. 704-0010)according to the manufacturer's instructions. Nunc Immuno MaxiSorp 96well flat bottom plates (Fisher Cat. No. DIS-971-030J) were coated withmouse anti-human CD105 (Southern Biotechnologies Cat. No. 9811-01) at1.5 μg/ml in PBS overnight at +4° C. The following day, 100 ng/ml humanCD105 (R&D Systems Cat. No. 1097-EN) in PBS/2% BSA was added to thepre-coated plate and incubated at room temperature for 1 hour. Afterwashing three times with PBST, a three-fold dilution series ofbiotinylated TRC205 antibody (10 μg/ml to 0.014 μg/ml) in PBST/2% BSAwas added in duplicate to the plate. Following incubation for 1 hour atroom temperature, the plate was washed three times with PBST and bindingof the biotinylated TRC205 antibody was detected using streptavidin-HRP(Sigma Cat. No. 55512) and TMB substrate (Sigma Cat. No. T0440). OD450nm values were measured on a Dynex MRX TCII plate reader. From thebinding curve, the EC50 value was calculated to be 46 ng/ml (FIG. 17).

The ability of human BMP9 to compete with the TRC205 antibody forbinding to human CD105 was determined by competition ELISA as follows:Nunc Immuno MaxiSorp 96 well flat bottom plates (Fisher Cat. No.DIS-971-030J) were coated with mouse anti-human CD105 (SouthernBiotechnologies Cat. No. 9811-01) at 1.5 μg/ml in PBS overnight at +4°C. The following day, 100 ng/ml human CD105 (R&D Systems Cat. No.1097-EN) in PBS/2% BSA was added to the pre-coated plate and incubatedat room temperature for 1 h. After washing three times with PBST, athree-fold dilution series of either human BMP9 (R&D Systems Cat. No.3209-BP-010) (413 nM to 0.56 nM), or unlabelled TRC205 antibody (40 nMto 0.05 nM) or a single concentration of an irrelevant antibody (66 nM)was mixed with a fixed concentration of biotinylated TRC205 antibody (55ng/ml) and added to the plate. Following incubation for 1 hour at roomtemperature, the plate was washed three times with PBST and binding ofthe biotinylated TRC205 antibody was detected using streptavidin-HRP(Sigma Cat. No. 55512) and TMB substrate (Sigma Cat. No. T0440). OD450nm values were measured on a Dynex MRX TCII plate reader. From theresultant competition curves (FIG. 18), the IC50 values were calculatedas 1.7 nM (0.26 μg/ml) and 14.6 nM (0.35 μg/ml) for TRC205 antibody andBMP9 respectively.

Example 13

A cell based assay was established to demonstrate that antibody TRC205inhibits the phosphorylation of SMAD 1/5/8 in human fibroblasts. A MesoScale Discovery (MSD) capture ELISA assay was developed to detectphosphorylated SMAD1 and total SMAD1 in protein extracts.

Fifty high density 96-well plates will be custom-coated by MSD using 500mg of a monoclonal anti-SMAD1 antibody (Santa Cruz #sc-81378). Twodifferent detection antibodies will be used to detect phosphorylatedSMAD1/5/8 (Cell Signaling Technology #9511) and total SMAD1 (CellSignaling Technology #9743). Detection will be conducted using therecommended MSD's protocol and buffers. In brief, fibroblasts will belifted with Accutase (Invitrogen #A1110501), washed 2× in PBS and platedin a 96-well plate at 2500 cells/well in 25 ml of media. After 3 hoursof serum starvation, antibodies will be added to the wells in 25 ml ofmedia. After 1 hour, 2.5 ml of BMP9 (2 ng/ml) will be added to eachwell. After 30 min, cells will be lyzed with 50 ml of MSD lysis buffer2× (150 mM NaCl, 20 mM Tris, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1% Triton™X-100, supplemented with phosphatase and protease inhibitors) and keptfrozen at −20 degrees Centigrade.

Forty (40) ml of lysate will be used for the MSD assay (per themanufacturer's instructions). MSD plates will be washed on an ELx405Select CW plate washer (from Biotek) and the signal will be read on aSector Imager 6000 (from Meso Scale Discovery).

The primary end point will be a change in the ratio of phosphorylatedSMAD 1 protein divided by total SMAD1 in response to BMP-9 stimulation,between fibroblasts treated with antibody TRC105 or antibody TRC205compared to fibroblasts treated with an antibody that does not bind tohuman endoglin.

Example 14

A phase 3 study will be conducted in which approximately 600 patientswith idiopathic pulmonary fibrosis will be randomly assigned to receiveeither oral pirfenidone (2403 mg per day) or an anti-endoglin antibodyfor 52 weeks. The primary end point will be a change in forced vitalcapacity (FVC) or death at week 52. Secondary end points include, forexample, the 6-minute walk distance, progression-free survival,extension of life, dyspnea, and/or death from any cause or fromidiopathic pulmonary fibrosis. Other assessments can include theoccurrence of adverse events between the two treatment arms.

Example 15

A Phase 3 study will be conducted in which approximately 100 patientsaged 3-25 years with neurofibromas and imaging evidence of tumorprogression. Patients will be randomized in a double-blinded fashion toreceive an endoglin antibody or placebo (phase A) and crossed over tothe opposite treatment arm at the time of tumor progression (phase B).Neurofibroma volumes will be measured with MRI, and progression wasdefined as ≥20% volume increase. Time to progression (TTP) in phase Awill be the primary endpoint, and the trial will be powered to detectwhether endoglin antibody doubled TTP compared with placebo. Toxicity,response, and quality of life will also be monitored.

While certain embodiments of the present application have been shown anddescribed herein, it will be obvious that such embodiments are providedby way of example only. Numerous variations, changes, and substitutionsmay occur to those skilled in the art without departing from theinvention; it should be understood that various alternatives to theembodiments described herein may be employed in practicing the methodsdescribed herein.

What is claimed is:
 1. A method of treating or inhibiting amyelofibrosis or a neurofibromatosis in a subject in need thereof,comprising administering to the subject a composition that comprises anantibody, or antigen-binding fragment thereof, that binds endoglin, thatcomprises a heavy chain variable region having an amino acid sequenceset forth as SEQ ID NO: 89 and a light chain variable region having anamino acid sequence set forth as SEQ ID NO: 93, and a pharmaceuticallyacceptable excipient, whereby the myelofibrosis or the neurofibromatosisis treated or inhibited.
 2. The method of claim 1, wherein theantigen-binding fragment is a Fab fragment, a Fab′ fragment, a F(ab′)₂fragment, an Fv fragment, an scFv fragment, or a single chain bindingpolypeptide.
 3. The method of claim 1, wherein the antibody, orantigen-binding fragment thereof, is further labeled with a therapeuticlabel.
 4. The method of claim 1, wherein the fibrosis is aneurofibromatosis that is a neurofibromatosis type 1 or aneurofibromatosis type
 2. 5. The method of claim 1, further comprisingadministering to the subject one or more fibrosis inhibitors.
 6. Themethod of claim 5, wherein the anti-endoglin antibody, orantigen-binding fragment and the one or more fibrosis inhibitors areadministered to the subject at the same site.
 7. The method of claim 5,wherein the anti-endoglin antibody, or antigen-binding fragment and theone or more fibrosis inhibitors are administered to the subject atdifferent sites.
 8. The method of claim 5, wherein the anti-endoglinantibody, or antigen-binding fragment and the one or more fibrosisinhibitors are administered to the subject sequentially.
 9. The methodof claim 5, wherein the anti-endoglin antibody, or antigen-bindingfragment and the one or more fibrosis inhibitors are administered to thesubject concurrently.
 10. The method of claim 1, wherein the antibody orantigen-binding fragment thereof is administered to the subject in anamount of about 0.01 mg/kg, about 0.05 mg/kg, about 0.1 mg/kg, about 0.5mg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 20 mg/kg,about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 60 mg/kg, about 70mg/kg, about 80 mg/kg, about 90 mg/kg, about 100 mg/kg, about 125 mg/kg,about 150 mg/kg, about 175 mg/kg, or about 200 mg/kg per subject.
 11. Amethod of treating or inhibiting a myelofibrosis or a neurofibromatosisin a subject in need thereof, comprising administering to the subject acomposition comprising an antibody, or antigen-binding fragment thereof,that binds endoglin, that comprises a heavy chain variable region havingan amino acid sequence set forth as SEQ ID NO: 89 with one or moremodifications selected from the group consisting of a substitution ofglycine (G) by alanine (A) or serine (S) at position 49; a substitutionof alanine (A) by isoleucine (I) at position 51; a substitution oflysine (K) by arginine (R) or glutamine (Q) at position 54; asubstitution of leucine (L) by valine (V) at position 81; and a lightchain variable region having an amino acid sequence set forth as SEQ IDNO: 93 with one or more modifications selected from the group consistingof a substitution of methionine (M) by leucine (L) at position 4; asubstitution of alanine (A) by valine (V) at position 19; a substitutionof threonine (T) by serine (S) at position 22; a substitution of alanine(A) by isoleucine (I) at position 48; and a substitution of threonine(T) by serine (S) at position 51; and a pharmaceutically acceptableexcipient, whereby the myelofibrosis or the neurofibromatosis is treatedor inhibited.
 12. The method of claim 11, wherein the antibody, orantigen-binding fragment thereof, comprises a heavy chain variableregion having an amino acid sequence set forth as SEQ ID NO: 88, 89, 90,91 or 92; and a light chain variable region having an amino acidsequence set forth as SEQ ID NO: 93, 94, 95, 96, 97, 100, 102, or 103.13. The method of claim 11, wherein the antibody, or antigen-bindingfragment thereof, comprises a heavy chain variable region having anamino acid sequence set forth as SEQ ID NO: 89, and a light chainvariable region having an amino acid sequence set forth as SEQ ID NO:94, 95, 96, 97, 100, 102, or
 103. 14. The method of claim 11, whereinthe fibrosis is a neurofibromatosis that is a neurofibromatosis type 1or a neurofibromatosis type
 2. 15. The method of claim 11, furthercomprising administering to the subject one or more fibrosis inhibitors.16. The method of claim 11, wherein the antibody or antigen-bindingfragment thereof is administered to the subject in an amount of about0.01 mg/kg, about 0.05 mg/kg, about 0.1 mg/kg, about 0.5 mg/kg, about 1mg/kg, about 5 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg,about 40 mg/kg, about 50 mg/kg, about 60 mg/kg, about 70 mg/kg, about 80mg/kg, about 90 mg/kg, about 100 mg/kg, about 125 mg/kg, about 150mg/kg, about 175 mg/kg, or about 200 mg/kg per subject.